Treatment of allodynia, hyperalgesia, spontaneous pain and phantom pain

ABSTRACT

The present invention relates to the use of Meteorin for the treatment of allodynia, hyperalgesia, spontaneous pain and phantom pain. In a preferred embodiment the disorder to be treated is allodynia, and hyperalgesia, more preferably allodynia including thermal and tactile allodynia.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/390,791 filed on 7 Oct., 2010, Denmark ApplicationNo. PA 2010 70423 filed on 1 Oct. 2010, and International PatentApplication No. PCT/DK2011/050369 filed on 30 Sep. 2011, the contents ofeach are hereby incorporated by reference.

All patent and non-patent references cited in the application, or in thepresent application, are also hereby incorporated by reference in theirentirety.

FIELD OF INVENTION

The present invention relates to the use of Meteorin for the treatmentof allodynia, hyperalgesia, spontaneous pain and phantom pain. In apreferred embodiment the disorder to be treated is allodynia, andhyperalgesia, more preferably allodynia including thermal and tactileallodynia. In another preferred embodiment the disorder is thermalhyperalgesia.

BACKGROUND OF INVENTION

Many therapies have been explored for the treatment of allodynia,hyperalgesia, spontaneous pain and phantom pain with varying degree ofsuccess, including non-steroidal anti-inflammatory drugs (NSAIDs),opioids, anticonvulsants, anti-arrhythmics, tricyclic antidepressantsand topical agents. Alternative approaches include anaesthetic blocks,epidural administration of steroids and neurosurgical lesions. However,all of the present therapies have modest efficacy in most patients andare palliative rather than curative and their side effects representsignificant limitations.

Hence, there is a high unmet need for therapies that treat allodynia,hyperalgesia, spontaneous pain and phantom pain effectively, preferablywith only minor side effects not affecting the general health of thepatients.

SUMMARY OF INVENTION

The present invention provides methods for treatment of allodynia,hyperalgesia, spontaneous pain and phantom pain. The methods useMeteorin protein, nucleotide sequences encoding Meteorin, expressionvectors containing the nucleotide sequence encoding Meteorin, cell linestransformed/transfected with the expression vector encoding Meteorin, orbiocompatible capsule delivering secreted Meteorin.

Thus, in a first aspect the present invention relates to an isolatedpolypeptide for use in a method of treatment of allodynia, hyperalgesia,spontaneous pain and/or phantom pain, said polypeptide comprising anamino acid sequence selected from the group consisting of:

-   -   i. The amino acid sequence of SEQ ID NO: 3;    -   ii. A biologically active sequence variant of the amino acid        sequence of SEQ ID NO:3, wherein the variant has at least 70%        sequence identity to SEQ ID NO:3; and    -   iii. A biologically active fragment of at least 50 contiguous        amino acids of i) or ii) wherein the fragment is at least 70%        identical to SEQ ID NO: 3.

The inventors have found that Meteorin is capable of alleviatingallodynia in animal models of both thermal and mechanical allodynia andspontaneous pain (weight bearing deficit). Importantly the animals didnot experience any weight loss or signs of toxicity over the duration ofthe experiment and no painful side effects were observed. The positiveeffects have been observed independently in several different models ofallodynia and spontaneous pain and using both systemic (subcutaneous)and local (intrathecal) administration

In a further aspect the invention relates to an isolated nucleic acidmolecule for use in a method of treatment of allodynia, hyperalgesia,spontaneous pain and/or phantom pain, said nucleic acid moleculecomprising a nucleic acid sequence encoding a polypeptide, saidpolypeptide comprising an amino acid sequence selected from the groupconsisting of:

-   -   i. The amino acid sequence of SEQ ID NO: 3;    -   ii. A biologically active sequence variant of the amino acid        sequence of SEQ ID NO:3, wherein the variant has at least 70%        sequence identity to SEQ ID NO:3; and    -   iii. A biologically active fragment of at least 50 contiguous        amino acids of i) or ii) wherein the fragment is at least 70%        identical to SEQ ID NO:3.

In a further aspect the invention relates to an expression vectorcomprising a nucleic acid molecule of the invention for use in a methodof treatment of allodynia, hyperalgesia, spontaneous pain and/or phantompain.

In a still further aspect the invention relates to an isolated host cellcomprising an expression vector according to the invention for use in amethod of treatment of allodynia, hyperalgesia, spontaneous pain and/orphantom pain. In particular the invention relates to host cells usefulfor cell based therapy, either naked cell based therapy or encapsulatedcell therapy for use in a method of treatment of allodynia,hyperalgesia, spontaneous pain and/or phantom pain.

In a further aspect the invention relates to an implantablebiocompatible capsule for use in a method of treatment of allodynia,hyperalgesia, spontaneous pain and/or phantom pain by delivery ofsecreted biologically active Meteorin to a subject, said capsulecomprising:

-   -   i. A biocompatible outer membrane and an inner core,    -   ii. Said inner core comprising cells according to the invention,    -   iii. Said cells comprising a vector according to the invention.

In a further aspect the invention relates to a composition comprising:

-   -   i. The isolated polypeptide according to the invention; or    -   ii. The isolated nucleic acid according to the invention; or    -   iii. The expression vector according to the invention; or    -   iv. The cell line according to the invention; or    -   v. An implantable biocompatible capsule according to the        invention;        for use in a method of treatment of allodynia, hyperalgesia,        spontaneous pain and/or phantom pain.

In a further aspect the invention relates to use of:

-   -   i. The isolated polypeptide according to the invention;    -   ii. The isolated nucleic acid according to the invention;    -   iii. The expression vector according to the invention;    -   iv. The cell line according to the invention; and    -   v. An implantable biocompatible capsule according to the        invention;        in the manufacture of a medicament for the treatment of        allodynia, hyperalgesia, spontaneous pain and/or phantom pain.

In a further aspect the invention relates to a method of treatment ofallodynia, hyperalgesia, spontaneous pain and/or phantom pain in asubject comprising administrating to said subject in need thereoftherapeutically effective amounts of the isolated polypeptide accordingto the invention.

DESCRIPTION OF DRAWINGS

FIG. 1. Ipsilateral hind paw withdrawal threshold to mechanicalstimulation with von Frey hairs following sciatic nerve injury. Notethat Meteorin treatment dose-dependently alleviates mechanicalallodynia. Arrows indicate treatment time points. The data are shown asmean±SEM and scoring was done blinded. *p<0.05.

FIG. 2. Ipsilateral hind paw response score to cold stimulationfollowing sciatic nerve injury. 0 is no response, 1 corresponds to astartle-like response seen in normal rats whereas 2 and 3 indicate mildand severe pain reactions. Note that Meteorin treatment dose-dependentlyalleviates cold allodynia. Arrows indicate treatment time points.Scoring was done blinded and data are shown as mean±SEM. *p<0.05.

FIG. 3. Body weight changes in sciatic nerve injured rats. All animalsgained weight normally throughout the study. Arrows indicate treatmenttime points. Scoring was done blinded and data are shown as mean±SEM.*p<0.05.

FIG. 4. Weight bearing deficits in rats after CCI (Chronic ConstrictionInjury). An incapacitance meter was used to assess the downward forceapplied by each hindlimb. Before surgery, there was no deficit as allanimals carried weight equally on both hind-limbs. After 12 days,immediately before treatment begins, ˜50 g more was put on thecontralateral limp compared to the ipsilateral limp. Note that Meteorintreatment alleviates the weight bearing deficits. Scoring was doneblinded and data are shown as mean±SEM. *p<0.05.

FIG. 5. Body weight changes in CCI rats. All animals gained weightnormally throughout the study. Arrows indicate treatment time points.Scoring was done blinded and data are shown as mean±SEM. *p<0.05.

FIG. 6. CLUSTAL W (1.82) multiple sequence alignment of Meteorin.

FIG. 6 a: alignment of Meteorin precursors from human (SEQ ID NO 2), rat(SEQ ID NO 8), and mouse (SEQ ID NO 5).

FIG. 6 b: alignment of mature Meteorin from human (SEQ ID NO 3), rat(SEQ ID NO 9), and mouse (SEQ ID NO 6).

FIG. 6 c: mature Meteorin, consensus sequence (SEQ ID NO 11) generatedfrom fully conserved residues in the human, mouse and rat sequences. Xrepresents any of the 21 naturally occurring amino acid encoded by DNA.

FIG. 7. Effect of Meteorin on mechanical hypersensitivity in CCI rats.Arrows indicate treatment days where animals were systemically injectedwith 0.1 mg/kg, 0.5 mg/kg or 1.8 mg/kg recombinant Meteorin or withvehicle as negative control. Rats were examined for altered nociceptionusing von Frey filaments and results expressed as means±SEM. * denotes asignificant difference (p<0.05) compared to vehicle treated animals.

FIG. 8. Effect of Meteorin on thermal hypersensitivity in CCI rats.Arrows indicate treatment days where animals were systemically injectedwith 0.1 mg/kg, 0.5 mg/kg or 1.8 mg/kg recombinant Meteorin or withvehicle as negative control. A Hargreaves device was used to assessthermal withdrawal latency and results expressed as means±SEM. * denotesa significant difference (p<0.05) between 1.8 mg/kg Meteorin and vehicletreated animals. # denotes a significant difference (p<0.05) between 0.5mg/kg Meteorin and vehicle treated animals. Note that Meteorinsignificantly and dose-dependently reduced thermal allodynia.

FIG. 9. Effect of Meteorin on differential weight bearing in CCI rats.Arrows indicate treatment days where animals were systemically injectedwith 0.1 mg/kg, 0.5 mg/kg or 1.8 mg/kg recombinant Meteorin or withvehicle as negative control. Differential weight bearing between theinjured and non-injured limb was determined using an incapacitance meterand expressed as % difference. Data are shown as means±SEM. * denotes asignificant difference (p<0.05) between 1.8 mg/kg Meteorin and vehicletreated animals. # denotes a significant difference (p<0.05) between 0.5mg/kg Meteorin and vehicle treated animals. $ denotes a significantdifference (p<0.05) between 0.1 mg/kg Meteorin and vehicle treatedanimals. Note that Meteorin significantly and dose-dependently reducedweight bearing deficits.

FIG. 10. Animal body weight during the CCI study. Arrows indicatetreatment days where animals were systemically injected with 0.1 mg/kg,0.5 mg/kg or 1.8 mg/kg recombinant Meteorin or with vehicle as negativecontrol. There were no changes in body weight in the Meteorin treatedgroups compared to vehicle.

FIG. 11. Meteorin in rat serum post systemic administration. Animalswere systemically injected with 0.1 mg/kg, 0.5 mg/kg or 1.8 mg/kgrecombinant Meteorin at Day 39 (t=0). Serum samples were collected at 2,6, 24 hr after injection and the concentration of Meteorin determinedusing ELISA. Meteorin was not detectable in serum samples from naïvecontrol rats.

FIG. 12. Effect of Meteorin on paw withdrawal threshold to mechanicalstimulation following ischemic sciatic nerve injury. Arrows indicatetime points for intreathecal injection. Data are shown as means±SEM. *indicates a significant difference (p<0.05) between vehicle and 6 μgMeteorin whereas # indicates a significant difference (p<0.05) betweenvehicle and 2 μg Meteorin.

FIG. 13. Effect of Meteorin on response to cold stimulation followingischemic sciatic nerve injury. Arrows indicate time points forintreathecal injection. Data are shown as means±SEM. * indicates asignificant difference (p<0.05) between vehicle and 6 μg Meteorinwhereas # indicates a significant difference (p<0.05) between vehicleand 2 μg Meteorin

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein “a biocompatible capsule” means that the capsule, uponimplantation in a host mammal, does not elicit a detrimental hostresponse sufficient to result in the rejection of the capsule or torender it inoperable, for example through degradation.

As used herein, a “coding sequence” is a polynucleotide sequence whichis transcribed and translated into a polypeptide.

As used herein, the term “Control sequence” refers to polynucleotidesequences which are necessary to effect the expression of coding andnon-coding sequences to which they are ligated. Control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence. In addition, “control sequences” refers tosequences which control the processing of the peptide encoded within thecoding sequence; these can include, but are not limited to sequencescontrolling secretion, protease cleavage, and glycosylation of thepeptide. The term “control sequences” is intended to include, at aminimum, components whose presence can influence expression, and canalso include additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

“Down regulation” of a promoter means the reduction in the expression ofthe product of transgene to a level, which may lead to a lack ofsignificant biological activity of the transgene product after in vivoimplantation. As used herein “a promoter not subject to down regulation”means a promoter, which, after in vivo implantation in a mammalian host,drives or continues to drive the expression of transgene at a levelwhich is biologically active.

As used herein, the term “expression vectors” refers to vectors that arecapable of directing the expression of genes to which they areoperatively-linked. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of plasmids.

As used herein, the terms “genetic modification” and “geneticengineering” refer to the stable or transient alteration of the genotypeof a cell by intentional introduction of exogenous DNA. DNA may besynthetic, or naturally derived, and may contain genes, portions ofgenes, or other useful DNA sequences. The term “genetic modification” isnot meant to include naturally occurring alterations such as that whichoccurs through natural viral activity, natural genetic recombination, orthe like.

As used herein “an immunoisolatory capsule” means that the capsule uponimplantation into a mammalian host minimizes the deleterious effects ofthe host's immune system on the cells within its core.

As used herein “long-term, stable expression of a biologically activecompound” means the continued production of a biologically activecompound at a level sufficient to maintain its useful biologicalactivity for periods greater than one month, preferably greater thanthree months and most preferably greater than six months.

By a “mammalian promoter” is intended a promoter capable of functioningin a mammalian cell.

Meteorin, as used herein, refers to polypeptides having the amino acidsequences of substantially purified Meteorin obtained from any species,particularly mammalian, including chimpanzee, bovine, ovine, porcine,murine, equine, and preferably human, from any source whether natural,synthetic, semi-synthetic, or recombinant. The term also refers tobiologically active fragments of Meteorin obtained from any of thesespecies, as well as to biologically active sequence variants of theseand to proteins subject to posttranslational modifications.

Growth factor characteristics as used herein define sequence-relatedfeatures similar to those of classical growth factors, which aresecreted proteins acting on a target cell through a receptor to causeone or more of the following responses in the target cell: growthincluding proliferation, differentiation, survival, regeneration,migration, regain of function, improvement of function trophic supportsuch as neurotrophic support.

As used herein, the term “operatively-linked” is intended to mean thatthe nucleotide sequence of interest is linked to the regulatorysequence(s) within a recombinant expression vector, in a manner thatallows for expression of the nucleotide sequence (e.g., in an in vitrotranscription/translation system or in a host cell when the vector isintroduced into the host cell).

As used herein, the term “regulatory sequence” is intended to includepromoters, enhancers and other expression control elements (e.g.,polyadenylation signals).

“Sequence identity”: A high level of sequence identity indicateslikelihood that the first sequence is derived from the second sequence.Amino acid sequence identity requires identical amino acid sequencesbetween two aligned sequences. Thus, a candidate sequence sharing 70%amino acid identity with a reference sequence, requires that, followingalignment, 70% of the amino acids in the candidate sequence areidentical to the corresponding amino acids in the reference sequence.Identity may be determined by aid of computer analysis, such as, withoutlimitations, the ClustalW computer alignment program (Higgins D.,Thompson J., Gibson T., Thompson J. D., Higgins D. G., Gibson T. J.,1994. CLUSTAL W: improving the sensitivity of progressive multiplesequence alignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680),and the default parameters suggested therein. The ClustalW software isavailable as a ClustalW WWW Service at the European BioinformaticsInstitute from http://www.ebi.ac.uk/clustalw. Using this program withits default settings, the mature (bioactive) part of a query and areference polypeptide are aligned. The number of fully conservedresidues are counted and divided by the length of the referencepolypeptide.

The ClustalW algorithm may similarly be used to align nucleotidesequences. Sequence identities may be calculated in a similar way asindicated for amino acid sequences.

The term “subject” used herein is taken to mean any mammal to whichMeteorin polypeptide or polynucleotide, therapeutic cells orbiocompatible capsules may be administered. Subjects specificallyintended for treatment with the method of the invention include humans,as well as nonhuman primates, sheep, horses, cattle, goats, pigs, dogs,cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice, as well asthe organs, tumors, and cells derived or originating from these hosts.

As used herein, the term “Transformation” refers to the insertion of anexogenous polynucleotide (i.e., a “transgene”) into a host cell. Theexogenous polynucleotide is integrated within the host genome.

“Treatment” can be performed in several different ways, includingcurative, ameliorating and as prophylaxis. Curative treatment generallyaims at curing a clinical condition, such as a disease or an infection,which is already present in the treated individual. Amelioratingtreatment generally means treating in order to improve, in anindividual, an existing clinical condition. Prophylactic treatmentgenerally aims at preventing a clinical condition or reducing the riskof contracting the condition or reducing the extent of the condition.

A treatment that can alter the underlying course of the disease. Byimpacting the actual disease process, “disease modification” therapy candelay, reverse or prevent progression of symptoms, or can change thelong term course of the disease

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. In the present specification, “plasmid” and “vector” can beused interchangeably as the plasmid is the most commonly used form ofvector. However, the invention is intended to include such other formsof expression vectors, such as viral vectors (e.g., replicationdefective retroviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions.

Allodynia

Allodynia, meaning “other pain”, is a pain due to a stimulus which doesnot normally provoke pain and can be either thermal ormechanical/tactile. It is pain from a stimulus that does not normallylead to the sensation of pain, and may occur after injury to a site.Allodynia is different from hyperalgesia and spontaneous pain, which isdescribed in the section “hyperalgesia” and “spontaneous pain”respectively.

There are different kinds or types of allodynia:

-   -   Mechanical allodynia (also known as tactile allodynia)        -   Static mechanical allodynia—pain in response to light            touch/pressure        -   Dynamic mechanical allodynia—pain in response to brushing    -   Thermal (heat or cold) allodynia—pain from normally mild skin        temperatures in the affected area

Allodynia is a clinical feature of many painful conditions, such asneuropathies, complex regional pain syndrome, postherpetic neuralgia,fibromyalgia, and migraine. Allodynia may also be caused by somepopulations of stem cells used to treat nerve damage including spinalcord injury. In a preferred embodiment of the present invention theallodynia to be treated is cold allodynia. In another preferredembodiment of the present invention the allodynia to be treated is heatallodynia.

The cell types involved in nociception and mechanical sensation are thecells responsible for allodynia. In healthy individuals, nociceptorssense information about cell stress or damage and temperature at theskin and transmit it to the spinal cord. The cell bodies of theseneurons lie in dorsal root ganglia, important structures located on bothsides of the spinal cord. The axons then pass through the dorsal horn tomake connections with secondary neurons. The secondary neurons crossover to the other (contralateral) side of the spinal cord and reachnuclei of the thalamus. From there, the information is carried throughone or more neurons to the somatosensory cortex of the brain.Mechanoreceptors follow the same general pathway. However, they do notcross over at the level of the spinal cord, but at the lower medullainstead. In addition, they are grouped in tracts that are spatiallydistinct from the nociceptive tracts.

Despite this anatomical separation, mechanoreceptors can influence theoutput of nociceptors by making connections with the same interneurons,the activation of which can reduce or completely eliminate the sensationof pain. Another way to modulate the transmission of pain information isvia descending fibers from the brain. These fibers act through differentinterneurons to block the transmission of information from thenociceptors to secondary neurons.

Both of these mechanisms for pain modulation have been implicated in thepathology of allodynia. Several studies suggest that injury to thespinal cord might lead to loss and re-organization of the nociceptors,mechanoreceptors and interneurons, leading to the transmission of paininformation by mechanoreceptors. A different study reports theappearance of descending fibers at the injury site. All of these changesultimately affect the circuitry inside the spinal cord, and the alteredbalance of signals probably leads to the intense sensation of painassociated with allodynia.

Different cell types have also been linked to allodynia. For example,there are reports that microglia in the thalamus might contribute toallodynia by changing the properties of the secondary nociceptors. Thesame effect is achieved in the spinal cord by the recruitment of immunesystem cells such as monocytes/macrophages and T lymphocytes.

As already mentioned, there are descending neurons that modulate theperception of pain. Many of these neurons originate in nuclei in thebrainstem and pass through the periaqueductal gray (PAG) area of themidbrain.

The body possesses an additional mechanism to control pain: the releaseof endogenous opioids, especially at the level of the PAG. There areneurons that release enkephalins, endorphins, and dynorphins at the PAG,and in this way modulate its ability to modulate pain perception. Otherneurons can release their endogenous opioids at the source of the pain,as well. If this occurs, the transmission of pain information from thenociceptors to the secondary neurons is blocked, and no pain is felt.Unfortunately, these endogenous mechanisms are often damaged andnonfunctional in people suffering from allodynia, so the application ofpharmaceuticals is needed.

Numerous compounds alleviate the pain from allodynia. Some are specificfor certain types of allodynia while others are general. They includenon-steroidal anti-inflammatory drugs (NSAIDs), opioids, and compoundstargeting different ion channels.

The present invention relates to the use of Meteorin for treatment ofallodynia. Preferably the allodynia to be treated is thermal allodynia.

As documented by example 4 full reversal to normal sensory function wasachieved in the majority of animals in the group receiving the highestdosage of Meteorin (1.8 mg/kg). It is thus conceivable that Meteorin canresult in substantially full reversal of allodynia in at least a subsetof the treated subjects. In a preferred embodiment, the treatmentresults in disease modification in at least a subset of the treatedsubjects.

Hyperalgesia

Hyperalgesia is an extreme response to a stimulus which is normallyperceived as painful. The stimulus can be mechanical/tactile or thermal.

Hyperalgesia is similar to other sorts of pain associated with nervedamage such as allodynia, and consequently may respond to standardtreatment for this condition as described in the section “allodynia”.

In one embodiment the present invention relates to the use of Meteorinfor treatment of hyperalgesia. Preferably the hyperalgesia to be treatedis thermal hyperalgesia. In one embodiment of the present invention thehyperalgesia to be treated is cold hyperalgesia. In another embodimentof the present invention the hyperalgesia to be treated is heathyperalgesia. As stated above substantially full reversal of normalsensory function was achieved in animals receiving the highest dosage ofMeteorin. It is thus conceivable that Meteorin can result in fullreversal of hyperalgesia in at least a subset of the treated subjects.In a preferred embodiment, the treatment results in disease modificationin at least a subset of the treated subjects.

Spontaneous Pain

Spontaneous pain is characterized by being pain occurring without anytrigger. The clinical symptoms of spontaneous pain include sensations ofpins and needles, shooting, burning, stabbing and paroxysmal (electricshock-like) pain sometimes associated with dysesthesia and/orparesthesia. Dysesthesia is defined as an unpleasant, abnormal sense oftouch, and it may be considered as a kind of pain occurringspontaneously. Paresthesia is defined as a sensation of tingling,pricking or numbness of a subjects skin with no apparent long-termphysical effect. Spontaneous pain seems likely to be caused byspontaneous activity of neurons in the afferent pathway.

In one embodiment the present invention relates to the use of meteorinfor treatment of spontaneous pain. It is thus conceivable that Meteorincan result in full reversal of spontaneous pain in at least a subset ofthe treated subjects. In a preferred embodiment, the treatment resultsin disease modification in at least a subset of the treated subjects.

Phantom Pain

Phantom pain sensations are described as perceptions that a subjectexperiences relating to a limb or an organ that is not physically partof the body. Phantom pain sensations are recorded most frequentlyfollowing the amputation of an arm or a leg, but may also occurfollowing the removal of a breast or an internal organ. The phantom painsensation varies from individual to individual. Phantom pain can beexperienced as sensations related to movement, touch, temperature,pressure and itchiness.

In one embodiment the present invention relates to the use of meteorinfor treatment of phantom pain.

Causes of Allodynia, and Hyperalgesia

Allodynia, hyperalgesia and in general hypersensitivity can arise from avariety of disorders, some of which are listed below.

Class Sub-type of cause Traumatic mechanical injury Entrapmentneuropathy Nerve transection Spinal cord injury Post-surgical painPhantom limb pain Scar formation Sciatica Metabolic or nutritionalAlcoholic neuropathy Pellagra Beriberi Burning foot syndrome ViralPost-herpetic neuralgia HIV/AIDS pain Neurotoxicity VincristineCisplatine Taxol Thallium Arsenic Radiation therapy Disease (non-viral)Diabetes Malignancies Multiple sclerosis Trigeminal neuralgiaGuillain-Barre syndrome Fabry's disease Tangier diseaseVasculitic/angiopathic Amyloid Idiopathic Ischaemia Thalamic syndromePost-stroke pain Neurotransmitter function Complex regional painsyndrome

Thus in one embodiment the invention relates to treatment of allodynia,hyperalgesia, or hypersensitivity in a subject diagnosed with a disorderlisted in the table above. Preferably, the invention relates totreatment of hypersensitivity in a subject diagnosed with painfuldiabetic neuropathy, post-herpetic neuralgia, or sciatica. Morepreferably, the invention relates to treatment of allodynia orhyperalgesia in a subject diagnosed with painful diabetic neuropathy,post-herpetic neuralgia, or sciatica. In an even more preferredembodiment, the invention relates to treatment of allodynia in a subjectdiagnosed with painful diabetic neuropathy, post-herpetic neuralgia, orsciatica.

Method of Treatment of Allodynia, Hyperalgesia, Spontaneous Pain and/orPhantom Pain

In one embodiment the present invention relates to the use of Meteorinfor the treatment of allodynia, hyperalgesia, spontaneous pain and/orphantom pain. In a more preferred embodiment the present inventionrelates to the use of Meteorin for the treatment of allodynia,hyperalgesia and/or spontaneous pain. In an even one embodiment thepresent invention relates to the use of Meteorin for treatment ofhyperalgesia and/or allodynia.

In a preferred embodiment the present invention relates to the use ofMeteorin for treatment of allodynia. In a more preferred embodiment thepresent invention relates to the use of Meteorin for the treatment ofmechanical allodynia. In an even more preferred embodiment the presentinvention relates to the use of Meteorin for treatment of thermalallodynia. In an even more preferred embodiment the present inventionrelates to the use of Meteorin for treatment of cold allodynia. In aneven more preferred embodiment the present invention relates to the useof Meteorin for treatment of heat allodynia.

In another preferred embodiment the present invention relates to the useof Meteorin for the treatment of spontaneous pain.

In another preferred embodiment the present invention relates to the useof Meteorin for the treatment of hyperalgesia. In a more preferredembodiment the present invention relates to the use of Meteorin for thetreatment of mechanical hyperalgesia. In an even more preferredembodiment the present invention relates to the use of Meteorin for thetreatment of thermal hyperalgesia. In an even more preferred embodimentthe present invention relates to the use of Meteorin for the treatmentof cold hyperalgesia. In an even more preferred embodiment the presentinvention relates to the use of Meteorin for treatment of heathyperalgesia.

The appended examples (example 4) demonstrate that the effect ofMeteorin is long-lasting in particular in view of the relatively shortserum half-life of Meteorin (FIG. 11). This indicates that Meteorin notonly alleviates the symptoms of hyperalgesia, hypersensitivity,allodynia and spontaneous pain, but that Meteorin may actually becapable of modifying the underlying disease or disorder. Thus, in oneembodiment, the treatment is a disease modifying treatment.

The examples also demonstrate that some of the tested subjectexperienced full reversal of their sensory dysfunction. Thus in oneembodiment, the treatment results in full reversal of sensorydysfunction, preferably full reversal of allodynia, more preferably fullreversal of tactile allodynia in at least a subset of the treatedsubjects. In another preferred embodiment, the treatment results insubstantially full reversal of hyperalgesia in at least a subset of thetreated subjects.

Treatment of Neuropathic Pain

Neuropathic pain is a category of pain that includes several forms ofchronic pain and which results from dysfunction of nervous rather thansomatic tissue. Neuropathic pain, that is pain deriving from dysfunctionof the central or peripheral nervous system, may also be a consequenceof damage to peripheral nerves or to regions of the central nervoussystem, may result from disease, or may be idiopathic. Symptoms ofneuropathic pain include sensations of burning, tingling, electricity,pins and needles, paresthesia, dysesthesia, stiffness, numbness in theextremities, feelings of bodily distortion, allodynia (pain evoked bystimulation that is normally innocuous), hyperalgesia (abnormalsensitivity to pain), hyperpathia (an exaggerated pain responsepersisting long after the pain stimuli cease), phantom pain, andspontaneous pain.

Current therapies for the management of neuropathic pain are of limitedbenefit to many patients, and involve undesirable side effects ordose-limiting toxicities. In addition, current therapies aresymptomatic, not disease modifying. Needs remain for improved therapiesfor the management and treatment of neuropathic pain, especially thosethat have the capacity to modify the disease.

In a series of animal studies the present inventors have observed thatadministration of dosages of Meteorin leads to long lasting improvementin tactile and thermal allodynia as well as spontaneous pain. In severalcases, the therapeutic effect is still detectable in the animals oneweek after administration of the last dosage. In other cases thetherapeutic effect is still detectable and significantly different fromcontrol treatment as long as two or even three weeks after the lastdosage.

In the observed cases, Meteorin polypeptide has been delivered assubcutaneous or intrathecal injections every second or third day for 9or 11 days. Meteorin is undetectable in the serum of animals 24 hoursafter subcutaneous injection. Therefore any build-up of Meteorin underthe observed administration schemes used is unlikely. The long lastingeffect of Meteorin may be caused by epigenetic changes or by repair ofthe nerve damages in the animals. Repair may be through regain offunction, neurogenesis or differentiation of neuronal precursors.

In any event it is highly surprising that a therapeutic effect can beobserved so long time after treatment cessation. In approved neuropathicpain drugs, such as gabapentin, serotonin-norepinephrine reuptakeinhibitors, tricyclic antidepressants, pain killers, cannabinoids, andopiods therapeutic therapeutic effect is not seen so long time afteradministration of the latest dosage. For example, in the case ofopioids, efficacy is contingent on the drug being present in bloodserum. When the blood serum level of the drug drops below a certainthreshold, no therapeutic effect is observed.

As the present inventors have demonstrated the Meteorin administered atlong dosage intervals is effective in treating different symptoms ofdifferent types neuropathic pain including thermal and tactile allodyniaand spontaneous pain, the present inventors contemplate that neuropathicpain in general can be treated by administering Meteorin polypeptide atrelatively long dosage intervals.

By a relatively long dosage interval is intended at least 2 days betweendosages, such as at least 3 days between dosages, for example 2 dosagesper week. More preferably the long dosages interval is at least oneweek, such as at least 2 weeks, more preferably at least 3 weeks, suchas at least 4 weeks, or at least one month.

Expressed in a different way the dosage intervals are so long thatfollowing one dosage of Meteorin polypeptide, the polypeptide is nolonger detectable in the serum of the subject to be treated when thenext dosage is administered. In another embodiment the blood serum levelis below 10 ng/mL, such as below 5 ng/mL, more preferably below 1 ng/mL,such as below 0.5 ng/mL, for example below 0.1 ng/mL.

In some embodiments, the long dosage range is preceded by more frequentinitial administration of Meteorin, e.g., twice daily, daily, once everytwo days, once every three days, or once every four days. This initialdosing schedule may be maintained e.g., for 2, 3, 4, 5, 6, 7, 9, 11, 14,21 days, or more. After completion of this dosing schedule, meteorin canbe administered less frequently, e.g., as described above.

Thus in one aspect, the invention relates to a method of treatingneuropathic pain in a human subject in need thereof comprisingadministering to the subject a therapeutically effective amount of aneurotrophic polypeptide comprising an amino acid sequence having atleast 70% identity to the amino acid sequence of SEQ ID NO: 3. whereinsaid administration is three times per week or more infrequently.

Preferably, the administration is weekly or more infrequentadministration. Even more preferably the administration is bi-weekly ormore infrequent administration.

In one embodiment the therapeutic effect of said treatment amelioratesat least one symptom of neuropathic pain for the entire period betweenpolypeptide administrations. The at least one symptom may be selectedfrom the group consisting of allodynia, hyperalgesia, spontaneous pain,phantom pain, sensations of burning, tingling, electricity, pins andneedles, paresthesia, dysesthesia, stiffness, numbness in theextremities, feelings of bodily distortion, and hyperpathia (anexaggerated pain response persisting long after the pain stimuli cease).Preferably the at least one symptom is selected from allodynia,hyperalgesia and spontaneous pain. More preferably allodynia.

Preferably said treatment does not maintain measurable levels of saidpolypeptide in the serum of said subject for the entire period betweenpolypeptide administrations. Preferably, the level of said polypeptidein the serum of said subject falls below 10 ng/mL, such as below 5ng/mL, more preferably below 1 ng/mL, such as below 0.5 ng/mL, forexample below 0.1 ng/mL between polypeptide administrations.

In another related aspect, the invention relates to a method of treatingneuropathic pain in a human subject in need thereof comprisingadministering to the subject a therapeutically effective amount of aneurotrophic polypeptide comprising an amino acid sequence having atleast 70% identity to the amino acid sequence of SEQ ID NO: 3, whereinsaid treatment does not maintain measurable levels of said polypeptidein the serum of said subject for the entire interval between polypeptideadministrations.

The invention also relates to use of the polypeptide of the invention insaid methods of treatment of neuropathic pain and to use of thepolypeptides of the invention in the manufacture of a medicament forsaid treatment of neuropathic pain.

Preferably the level of said polypeptide in the serum of said subjectfalls below 10 ng/mL, such as below 5 ng/mL, more preferably below 1ng/mL, such as below 0.5 ng/mL, for example below 0.1 ng/mL betweenpolypeptide administrations.

For these aspects of the invention relating to treatment of neuropathicpain using long dosage intervals, the neurotrophic polypeptidepreferably has at least 85% sequence identity to the amino acid sequenceof SEQ ID NO: 3, more preferably at least 90%, more preferably at least95%, more preferably at least 98%.

In one embodiment the neurotrophic polypeptide comprises the consensussequence of SEQ ID NO:11.

Preferably the neurotrophic polypeptide has cysteine residues atpositions 7, 28, 59, 95, 148, 151, 161, 219, 243, and 265 relative tothe amino acid sequence of SEQ ID NO:3.

Meteorin

The present invention relates to the use of polypeptides beingidentified as Meteorin protein and polynucleotides encoding saidprotein, in the treatment of allodynia, hyperalgesia, spontaneous painand/or phantom pain. The delivery is in one embodiment contemplated tobe by use of a capsule for delivery of a secreted biologically activeMeteorin and/or a homologue thereof to a subject. The Meteorin proteinhas been identified in human beings (SEQ ID No. 2), mouse (SEQ ID No.5), and rat (SEQ ID No. 8) and a variety of other species.

Human Meteorin exists as a 293 amino acid precursor, which can beprocessed to give rise to at least one biologically active peptide.Meteorin is expressed at high levels in the nervous system and the eye,and in particular subregions of the brain. The mouse (SEQ ID No 5) andrat (SEQ ID No 8) Meteorin precursors consist of 291 amino acids, andthe % identities with the human Meteorin protein (SEQ ID NO: 2) are 80.3and 80.2, respectively (See FIG. 6).

Human Meteorin contains an N-terminal signal peptide sequence of 23amino acids, which is cleaved at the sequence motif ARA-GY. This signalpeptide cleavage site is predicted by the SignalP method. The N-terminalof mouse Meteorin has been has been verified by N-terminal sequencing(Jørgensen et al., Characterization of meteorin—An evolutionaryconserved neurotrophic factor, J mol Neurosci 2009 September; 39 (1-2):104-116).

Table 1 shows the % sequence identity between full length human Meteorinversus mouse and rat sequences. See alignment in FIG. 6 a.

Sequence % id human — mouse 80.3 rat 80.2

Table 2 shows the % sequence identity between human Meteorin versusmouse and rat sequences after removal of N-terminal signal peptide. Seealignment in FIG. 6 b.

Sequence % id human — mouse 81.9 rat 79.6

Based on the fully conserved residues, a consensus sequence for matureMeteorin can be derived (FIG. 6 c), wherein X is independently selectedfrom any of the 21 naturally occurring amino acid encoded by DNA. In apreferred embodiment a variant Meteorin comprises the consensussequence.

The therapeutic effect of Meteorin may be mediated through aneurotrophic effect, an effect on growth including proliferation,regeneration, regain of function, improvement of function, survival,migration, and/or differentiation of targeted cells.

One biological function of Meteorin is the ability to induce neuriteoutgrowth in dissociated dorsal root ganglia (DRG) cultures as describedin Jorgensen et al., Characterization of meteorin—An evolutionaryconserved neurotrophic factor, J mol Neurosci 2009 September; 39 (1-2):104-116 and Nishino et al., “Meteorin: a secret3ed protein thatregulates glial cell differentitaion and promotes axonal extension”,EMBO J., 23(9):1998-2008 (2004).

Due to the high conservation of the cysteines, it is expected that theseresidues play an important role in the secondary and tertiary structureof the bioactive protein. One or more of the cysteines may participatein the formation of intra- and/or intermolecular cystin-bridges.

It has been demonstrated that Meteorin has a stimulating effect on thepercentage of neurons generated by a human neural stem cell line (hNS1,formerly called HNSC. 100) and Meteorin also has a stimulating effect ongeneration of neurons in a primary culture of rat striatal cells (see WO2005/095450).

Administration and Formulation

Meteorin polypeptides may be administered in any manner, which ismedically acceptable. This may include injections, by parenteral routessuch as intravenous, intravascular, intraarterial, subcutaneous,intramuscular, intratumor, intraperitoneal, intraventricular,intraepidural, intrathecal, intracerebroventricular, intercerebral, orothers as well as nasal, or topical. Slow release administration is alsospecifically included in the invention, by such means as depotinjections or erodible implants.

Administration of Meteorin according to this invention may be achievedusing any suitable delivery means, including:

injection, either subcutaneously, intravenously, intra-arterially,intramuscularly, intrathecally or to other suitable site;

pump (see, e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer,41:1270 (1993); Cancer Research, 44:1698 (1984), incorporated herein byreference),

microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and5,084,350, herein incorporated by reference),

slow release polymer implants (see, e.g., Sabel, U.S. Pat. No.4,883,666, incorporated herein by reference),

encapsulated cells (see, “Biocompatible capsules”),

unencapsulated cell grafts (see, e.g., U.S. Pat. Nos. 5,082,670 and5,618,531, each incorporated herein by reference); and

inhalation.

Administration may be by periodic injections of a bolus of thepreparation, or may be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.,an IV bag) or internal (e.g., a bioerodable implant, a bioartificialorgan, a biocompatible capsule of Meteorin production cells, or a colonyof implanted Meteorin production cells). See, e.g., U.S. Pat. Nos.4,407,957, 5,798,113, and 5,800,828, each incorporated herein byreference.

Localised delivery may be by such means as delivery via a catheter toone or more arteries. In one embodiment of the present inventionlocalised delivery comprises delivery using encapsulated cells (asdescribed in the section “biocompatible capsule”). A further type oflocalised delivery comprises local delivery of gene therapy vectors,which are normally injected.

In a preferred embodiment of the present invention the administration isparenteral injection, preferably subcutaneous injection or intrathecalinjection.

Whilst it is possible for the compounds of the present invention to beadministered as the raw chemical, it is preferred to present them in theform of a pharmaceutical formulation. The pharmaceutical formulationsmay be prepared by conventional techniques, e.g. as described inRemington: The Science and Practice of Pharmacy 2005, Lippincott,Williams & Wilkins.

The term “pharmaceutically acceptable carrier” means one or more organicor inorganic ingredients, natural or synthetic, with which Meteorinpolypeptide is combined to facilitate its application. A suitablecarrier includes sterile saline although other aqueous and non-aqueousisotonic sterile solutions and sterile suspensions known to bepharmaceutically acceptable are known to those of ordinary skill in theart.

The compounds of the present invention may be formulated for parenteraladministration and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containers,optionally with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, for example solutions in aqueous polyethylene glycol. Examplesof oily or non-aqueous carriers, diluents, solvents or vehicles includepropylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil),and injectable organic esters (e.g., ethyl oleate), and may containagents such as preserving, wetting, emulsifying or suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilisation from solution for constitution beforeuse with a suitable vehicle, e.g., sterile, pyrogen-free water.

An “effective amount” refers to that amount which is capable ofameliorating or delaying progression of the diseased, degenerative ordamaged condition. An effective amount can be determined on anindividual basis and will be based, in part, on consideration of thesymptoms to be treated and results sought. An effective amount can bedetermined by one of ordinary skill in the art employing such factorsand using no more than routine experimentation.

A liposome system may be any variety of unilamellar vesicles,multilamellar vesicles, or stable plurilamellar vesicles, and may beprepared and administered according to methods well known to those ofskill in the art, for example in accordance with the teachings of U.S.Pat. Nos. 5,169,637, 4,762,915, 5,000,958 or 5,185,154. In addition, itmay be desirable to express the novel polypeptides of this invention, aswell as other selected polypeptides, as lipoproteins, in order toenhance their binding to liposomes. A recombinant Meteorin protein ispurified, for example, from CHO cells by immunoaffinity chromatographyor any other convenient method, then mixed with liposomes andincorporated into them at high efficiency. The liposome-encapsulatedprotein may be tested in vitro for any effect on stimulating cellgrowth.

Where slow-release administration of a Meteorin polypeptide is desiredin a formulation with release characteristics suitable for the treatmentof any disease or disorder requiring administration of a Meteorinpolypeptide, microencapsulation of a Meteorin polypeptide iscontemplated. Microencapsulation of recombinant proteins for sustainedrelease has been successfully performed with human growth hormone(rhGH), interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson etal., Nat. Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223(1993); Hora et al., Bio/Technology, 8:755-758 (1990); Cleland, “Designand Production of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

The slow-release formulations of these proteins were developed usingpoly-lactic-coglycolic acid (PLGA) polymer due to its biocompatibilityand wide range of biodegradable properties. The degradation products ofPLGA, lactic and glycolic acids, can be cleared quickly within the humanbody. Moreover, the degradability of this polymer can be adjusted frommonths to years depending on its molecular weight and composition.Lewis, “Controlled release of bioactive agents from lactide/glycolidepolymer,” in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers asDrug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.

In one embodiment of the present invention a composition comprisingMeteorin is contemplated. The composition may comprise an isolatedpolypeptide as described herein, an isolated nucleic acid as describedherein, a Meteorin encoding expression vector as described herein, acell line expressing Meteorin as described herein or a biocompatiblecapsule secreting Meteorin as described herein.

Dosages

Various dosing regimes for systemic administration are contemplated. Inone embodiment, methods of administering to a subject a formulationcomprising a Meteorin polypeptide include administering Meteorin at adosage of between 1 μg/kg and 10,000 μg/kg body weight of the subject,per dose. In another embodiment, the dosage is between 1 μg/kg and 7,500μg/kg body weight of the subject, per dose. In a further embodiment, thedosage is between 1 μg/kg and 5,000 μg/kg body weight of the subject,per dose. In a different embodiment, the dosage is between 1 μg/kg and2,000 μg/kg body weight of the subject, per dose. In yet anotherembodiment, the dosage is between 1 μg/kg and 1,000 μg/kg body weight ofthe subject, per dose. In yet another embodiment, the dosage is between1 μg/kg and 700 μg/kg body weight of the subject, per dose. In a morepreferable embodiment, the dosage is between 5 μg/kg and 500 μg/kg bodyweight of the subject, per dose. In a most preferable embodiment, thedosage is between 10 μg/kg and 100 μg/kg body weight of the subject, perdose. In a preferred embodiment the subject to be treated is human.

Guidance as to particular dosages and methods of delivery is provided inthe literature; see, for example, WO 02/78730 and WO 07/100,898.Guidance to the calculation of the human equivalent dosages based ondosages used in animal experiments is provided in Reagan-Shaw et al.,FASEB J, 22, 659-661 (2007).

The dose administered must be carefully adjusted to the age, weight andcondition of the individual being treated, as well as the route ofadministration, dosage form and regimen, and the result desired, and theexact dosage should be determined by the practitioner.

In one embodiment of the present invention the administration isrepeated daily. In another embodiment the administration is repeated atleast 1-3 times weekly, such as 2-5 times weekly, such as 3-6 timesweekly, once every three days, once every four days, once every fivedays, once every six days, or once every 7 days.

In other embodiments, meteorin is administered at relatively long dosageinterval. A relatively long dosage interval is intended to include atleast 2 days between dosages, such as at least 3 days between dosages,for example 2 dosages per week. More preferably the long dosagesintervals is at least one week, such as at least 2 weeks, morepreferably at least 3 weeks, such as at least 4 weeks, or at least onemonth.

Expressed in a different way the dosage intervals are so long thatfollowing one dosage of Meteorin polypeptide, the polypeptide is nolonger detectable in the serum of the subject to be treated when thenext dosage is administered. In another embodiment the blood serum levelis below 10 ng/mL, such as below 5 ng/mL, more preferably below 1 ng/mL,such as below 0.5 ng/mL, for example below 0.1 ng/mL.

In some embodiments, the initial administration of Meteorin is, e.g.,twice daily, daily, once every two days, once every three days, or onceevery four days. This dosing schedule may be maintained e.g., for 2, 3,4, 5, 6, 7, 9, 11, 14, 21 days, or more. After completion of this dosingschedule, meteorin can be administered less frequently, e.g., asdescribed above.

Meteorin Polypeptides

In addition to full-length Meteorin, substantially full-length Meteorin,and to pro-Meteorin, the present invention provides for biologicallyactive variants of the polypeptides. A Meteorin polypeptide or fragmentis biologically active if it exhibits a biological activity of naturallyoccurring Meteorin as described herein, such as being neurotrophic. Itis to be understood that the invention relates to Meteorin as hereindefined.

The invention relates to an isolated polypeptide molecule for use in amethod of treatment of allodynia, hyperalgesia, spontaneous pain and/orphantom pain, said polypeptide comprising an amino acid sequenceselected from the group consisting of:

a) the amino acid sequence selected from the group consisting of SEQ IDNo. 3, 6 and 9;b) a biologically active sequence variant of the amino acid sequenceselected from the group consisting of SEQ ID No. 3, 6 and 9, wherein thevariant has at least 70% sequence identity to said SEQ ID No.; andc) a biologically active fragment of at least 50 contiguous amino acidsof any of a) or b) wherein the fragment is at least 70% identical tosaid SEQ ID NO.

In one embodiment the invention relates to an isolated polypeptideselected from the group consisting of:

-   -   i) AA₃₀-AA₂₈₈ of SEQ ID No 2, and polypeptides having from one        to five extra amino acids from the native sequence in one or        both ends, up to AA₂₅-AA₂₉₃ of SEQ ID No 2;    -   ii) AA₂₈-AA₂₈₆ of SEQ ID No 8 and polypeptides having from one        to five extra amino acids from the native sequence in one or        both ends, up to AA₂₃-AA₂₉₁ of SEQ ID No 8;    -   iii) AA₃₁-AA₂₈₉ of SEQ ID No 5 and polypeptides having from one        to five extra amino acids from the native sequence in one or        both ends, up to AA₂₆-AA₂₉₄ of SEQ ID No 5; and    -   iv) variants of said polypeptides, wherein any amino acid        specified in the chosen sequence is changed to a different amino        acid, provided that no more than 20 of the amino acid residues        in the sequence are so changed.

Biological activity preferably is neurotrophic activity.Neurotrophically active variants may be defined with reference to one ormore of the other in vitro and/or in vivo neurotrophic assays describedabove in WO 2005/095450, in particular the DRG assay.

A preferred biological activity is the ability to elicit substantiallythe same response as in the DRG assay described in Jorgensen et al.,Characterization of meteorin—An evolutionary conserved neurotrophicfactor, J mol Neurosci 2009 September; 39 (1-2): 104-116. In this assayDRG cells are grown in the presence of full length human Meteorin codingsequence (SEQ ID NO 3). By substantially the same response in the DRGassay is intended that the neurite outgrowth from DRG cells is at least20% of the number obtained in the DRG assay described in Jorgensen etal., Characterization of meteorin—An evolutionary conserved neurotrophicfactor, J mol Neurosci 2009 September; 39 (1-2): 104-116, morepreferably at least 30%, more preferably at least 40%, more preferablyat least 50%, more preferably at least 60%, more preferably at least70%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%. The biologicalactivity of a fragment or variant of Meteorin may also be higher thanthat of the naturally occurring Meteorin (SEQ ID NO 3).

Variants can differ from naturally occurring Meteorin in amino acidsequence or in ways that do not involve sequence, or in both ways.Variants in amino acid sequence (“sequence variants”) are produced whenone or more amino acids in naturally occurring Meteorin is substitutedwith a different natural amino acid, an amino acid derivative ornon-native amino acid. Particularly preferred variants include naturallyoccurring Meteorin, or biologically active fragments of naturallyoccurring Meteorin, whose sequences differ from the wild type sequenceby one or more conservative and/or semi-conservative amino acidsubstitutions, which typically have minimal influence on the secondaryand tertiary structure and hydrophobic nature of the protein or peptide.Variants may also have sequences, which differ by one or morenon-conservative amino acid substitutions, deletions or insertions,which do not abolish the Meteorin biological activity. The Clustal Walignment in FIG. 6 can be used to predict which amino acid residues canbe substituted without substantially affecting the biological activityof the protein. In a preferred embodiment a variant Meteorin sequencecomprises the consensus sequence having SEQ ID NO 11.

Substitutions within the following group (Clustal W, ‘strong’conservation group) are to be regarded as conservative substitutionswithin the meaning of the present invention

-   -   S,T,A; N,E,Q,K; N,H,Q,K; N,D,E,Q; Q,H,R,K; M,I,L,V; M,I,L,F;        H,Y; F,Y,W.

Substitutions within the following group (Clustal W, ‘weak’ conservationgroup) are to be regarded as semi-conservative substitutions within themeaning of the present invention

-   -   C,S,A; A,T,V; S,A,G; S,T,N,K; S,T,P,A; S,G,N,D; S,N,D,E,Q,K;        N,D,E,Q,H,K; N,E,Q,H,R,K; V,L,I,M; H,F,Y.

Other variants within the invention are those with modifications whichincrease peptide stability. Such variants may contain, for example, oneor more nonpeptide bonds (which replace the peptide bonds) in thepeptide sequence. Also included are: variants that include residuesother than naturally occurring L-amino acids, such as D-amino acids ornon-naturally occurring or synthetic amino acids such as beta or gammaamino acids and cyclic variants. Incorporation of D-amino acids insteadof L-amino acids into the polypeptide may increase its resistance toproteases. See, e.g., U.S. Pat. No. 5,219,990. Splice variants arespecifically included in the invention.

When the result of a given substitution cannot be predicted withcertainty, the derivatives may be readily assayed according to themethods disclosed herein to determine the presence or absence ofneurotrophic activity, preferably using the DRG assay described inJorgensen et al., Characterization of meteorin—An evolutionary conservedneurotrophic factor, J mol Neurosci 2009 September; 39 (1-2): 104-116.

In one embodiment, the polypeptide is a naturally occurring allelicvariant of the sequence selected from the group consisting of SEQ ID No.3, 6 and 9. This polypeptide may comprise an amino acid sequence that isthe translation of a nucleic acid sequence differing by a singlenucleotide from a nucleic acid sequence selected from the groupconsisting of SEQ ID No. 1, 4 and 7.

A variant polypeptide as described herein, in one embodiment comprises apolypeptide wherein any amino acid specified in the chosen sequence ischanged to provide a conservative substitution.

Variants within the scope of the invention in one embodiment includeproteins and peptides with amino acid sequences having at least 70percent identity with human, murine or rat Meteorin (SEQ ID NO: 3, 6,and 9). More preferably the sequence identity is at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 95%, more preferably at least98%.

In a preferred embodiment the sequence identity of the variant Meteorinis determined with reference to a human Meteorin polypeptide (SEQ ID No3).

In one embodiment, the variants include proteins comprising an aminoacid sequence having at least 70% sequence identity to SEQ ID NO 3, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least95%, more preferably at least 98%.

In one embodiment, preferred variants include proteins comprising anamino acid sequence having at least 70% sequence identity to SEQ ID NO6, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95%, more preferably at least 98%.

In one embodiment, preferred variants include proteins comprising anamino acid sequence having at least 70% sequence identity to SEQ ID NO9, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 95%, more preferably at least 98%.

In one embodiment, preferred variants of Meteorin include proteinscomprising 50-270 amino acids, more preferably 75-270 amino acids, morepreferably 90-270 amino acids, more preferably 100-270 amino acids, morepreferably 125-270 amino acids, more preferably 150-270 amino acids,more preferably 175-270 amino acids, more preferably 200-270 aminoacids, more preferably 225-270 amino acids, more preferably 250-270amino acids.

In one embodiment, a variant Meteorin at corresponding positionscomprises the residues marked in FIG. 6 as fully conserved (*), morepreferably a variant Meteorin also comprises at corresponding positionsthe residues marked in FIG. 6 as strongly conserved (: stronglyconserved groups include: S,T,A; N,E,Q,K; N,H,Q,K; N,D,E,Q; Q,H,R,K;M,I,L,V; M,I,L,F; H,Y; F,Y,W), more preferably a variant Meteorin alsocomprises at corresponding positions the residues marked in FIG. 6 asless conserved (. less conserved groups include: C,S,A; A,T,V; S,A,G;S,T,N,K; S,T,P,A; S,G,N,D; S,N,D,E,Q,K; N,D,E,Q,H,K; N,E,Q,H,R,K;V,L,I,M; H,F,Y). In particular, it is contemplated that the conservedcysteines must be located at corresponding positions in a variantMeteorin. Thus in one embodiment, a variant Meteorin sequence hascysteine residues at positions 7, 28, 59, 95, 148, 151, 161, 219, 243,and 265 relative to the amino acid sequence of SEQ ID NO: 3.

In one embodiment the neurotrophic polypeptide comprises the consensussequence of SEQ ID NO:11. The consensus sequence comprises the aminoacid residues conserved in human, mouse and rat meteorin as shown inFIG. 6. Preferably the neurotrophic polypeptide has cysteine residues atpositions 7, 28, 59, 95, 148, 151, 161, 219, 243, and 265 relative tothe amino acid sequence of SEQ ID NO:3.

Non-sequence modifications may include, for example, in vivo or in vitrochemical derivatisation of portions of naturally occurring Meteorin, aswell as acetylation, methylation, phosphorylation, carboxylation,PEG-ylation, or glycosylation. Just as it is possible to replacesubstituents of the protein, it is also possible to substitutefunctional groups, which are bound to the protein with groupscharacterized by similar features. Such modifications do not alterprimary sequence. These will initially be conservative, i.e., thereplacement group will have approximately the same size, shape,hydrophobicity and charge as the original group.

Many amino acids, including the terminal amino acids, may be modified ina given polypeptide, either by natural processes such as glycosylationand other post-translational modifications, or by chemical modificationtechniques which are well known in the art. Among the knownmodifications which may be present in polypeptides of the presentinvention are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a polynucleotide orpolynucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycation, glycosylation, GPI anchorformation, hydroxylation, iodination, methylation, myristoylation,oxidation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.

Such modifications are well known to those of skill and have beendescribed in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as, for instance, I. E. Creighton, Proteins-Structure and MolecularProperties, 2nd Ed., W. H. Freeman and Company, New York, 1993. Manydetailed reviews are available on this subject, such as, for example,those provided by Wold, F., in Posttranslational Covalent Modificationof Proteins, B. C. Johnson, Ed., Academic Press, New York, pp 1-12,1983; Seifter et al., Meth. Enzymol. 182: 626-646, 1990 and Rattan etal., Protein Synthesis: Posttranslational Modifications and Aging, Ann.N.Y. Acad. Sci. 663: 48-62, 1992.

In addition, the protein may comprise a protein tag to allow subsequentpurification and optionally removal of the tag using an endopeptidase.The tag may also comprise a protease cleavage site to facilitatesubsequent removal of the tag. Non-limiting examples of affinity tagsinclude a polyhis tag, a GST tag, a HA tag, a Flag tag, a C-myc tag, aHSV tag, a V5 tag, a maltose binding protein tag, a cellulose bindingdomain tag. Preferably for production and purification, the tag is apolyhistag. Preferably, the tag is in the C-terminal portion of theprotein.

The native signal sequence of Meteorin may also be replaced in order toincrease secretion of the protein in recombinant production in othermammalian cell types.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to proteolyticprocessing, almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell's posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, glycosylation often doesnot occur in bacterial hosts such as E. coli. Accordingly, whenglycosylation is desired, a polypeptide should be expressed in aglycosylating host, generally a eukaryotic cell. Insect cells oftencarry out the same posttranslational glycosylations as mammalian cellsand, for this reason, insect cell expression systems have been developedto efficiently express mammalian proteins having native patterns ofglycosylation, inter alia. Similar considerations apply to othermodifications.

It will be appreciated that the same type of modification may be presentto the same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

Meteorin Nucleotide Sequences

The invention provides medical use of genomic DNA and cDNA coding forMeteorin, including for example the human cDNA nucleotide sequence (SEQID No. 1 and 10), the mouse cDNA sequences (SEQ ID NO 4) and rat cDNAsequences (SEQ ID No. 7).

Variants of these sequences are also included within the scope of thepresent invention.

The invention relates to an isolated nucleic acid molecule for use in amethod of treatment of allodynia, hyperalgesia, spontaneous pain and/orphantom pain, said nucleic acid molecule comprising a nucleic acidsequence encoding a polypeptide, said polypeptide comprising an aminoacid sequence selected from the group consisting of:

-   -   i. The amino acid sequence of SEQ ID NO: 3;    -   ii. A biologically active sequence variant of the amino acid        sequence of SEQ ID NO:3, wherein the variant has at least 70%        sequence identity to SEQ ID NO:3; and    -   iii. A biologically active fragment of at least 50 contiguous        amino acids of i) or ii) wherein the fragment is at least 70%        identical to SEQ ID NO: 3.

In one embodiment the invention relates to an isolated nucleic acidmolecule for use in a method of treatment of allodynia, hyperalgesia,spontaneous pain and/or phantom pain encoding a polypeptide, saidpolypeptide comprising an amino acid sequence selected from the groupconsisting of:

-   -   i) AA₃₀-AA₂₈₈ of SEQ ID No 2, and polypeptides having from one        to five extra amino acids from the native sequence in one or        both ends, up to AA₂₅-AA₂₉₃ of SEQ ID No 2;    -   ii) AA₂₈-AA₂₈₆ of SEQ ID No 8 and polypeptides having from one        to five extra amino acids from the native sequence in one or        both ends, up to AA₂₃-AA₂₉₁ of SEQ ID No 8;    -   iii) AA₃₁-AA₂₈₉ of SEQ ID No 5 and polypeptides having from one        to five extra amino acids from the native sequence in one or        both ends, up to AA₂₆-AA₂₉₄ of SEQ ID No 5; and    -   iv) variants of said polypeptides, wherein any amino acid        specified in the chosen sequence is changed to a different amino        acid, provided that no more than 20 of the amino acid residues        in the sequence are so changed.

The nucleic acid molecule may comprise the nucleotide sequence of anaturally occurring allelic nucleic acid variant.

The nucleic acid molecule of the invention may encode a variantpolypeptide, wherein the variant polypeptide has the polypeptidesequence of a naturally occurring polypeptide variant.

In one embodiment the nucleic acid molecule differs by a singlenucleotide from a nucleic acid sequence selected from the groupconsisting of SEQ ID No. 1, 4, 7 and 10.

Preferably the encoded polypeptide has at least 60% sequence identity toa sequence selected from the group consisting of SEQ ID No. 3 preferablyat least 65% sequence identity, more preferably at least 70% sequenceidentity, more preferably, 75% sequence identity, more preferably atleast 80% sequence identity, more preferably at least 85% sequenceidentity, more preferably at least 90% sequence identity, morepreferably at least 95% sequence identity, more preferably at least 98%sequence identity, more preferably wherein the polypeptide has asequence selected from the group consisting of said SEQ ID No.s. Saidsequences constitute human Meteorin.

In a preferred embodiment, the encoded polypeptide comprises theconsensus sequence having SEQ ID NO:11.

In a preferred embodiment the encoded polypeptide has at least 70%sequence identity to SEQ ID No. 3, more preferably at least 75%, morepreferably at least 80%, more preferably at least 95%, more preferablyat least 98%, more preferably wherein said polypeptide has the sequenceof SEQ ID No. 3.

In one aspect the nucleic acid molecule comprises a nucleotide sequenceselected from the group consisting of

a) the nucleotide sequence selected from the group consisting of SEQ IDNo. 1, 4, 7 and 10;b) a nucleotide sequence having at least 70% sequence identity to anucleotide sequence selected from the group consisting of SEQ ID No. 1,4, 7 and 10; andc) a nucleic acid sequence of at least 150 contiguous nucleotides of asequence selected from the group consisting of SEQ ID No. 1, 4, 7 and10;

In one embodiment, the isolated polynucleotide of the invention has atleast 60, more preferably at least 65%, more preferably at least 70%,more preferably at least 75%, more preferably at least 80%, preferablyat least 85%, more preferred at least 90%, more preferred at least 95%,more preferred at least 98% sequence identity to the polynucleotidesequence presented as SEQ ID NO: 1.

In one preferred embodiment, the isolated polynucleotide of theinvention has at least 50%, preferably at least 60%, more preferably atleast 70%, more preferably at least 75%, more preferably at least 80%,preferably at least 85%, more preferred at least 90%, more preferred atleast 95%, more preferred at least 98% sequence identity to apolynucleotide sequence presented as SEQ ID NO: 10.

In one embodiment, preferred isolated polynucleotide variants of theinvention comprises 150-900 nucleic acids, more preferably 175-900nucleic acids, more preferably 200-900 nucleic acids, more preferably225-900 nucleic acids, more preferably 250-900 nucleic acids, morepreferably 300-900 nucleic acids, more preferably 350-900 nucleic acids,more preferably 400-900 nucleic acids, more preferably 450-900 nucleicacids, more preferably 500-900 nucleic acids, more preferably 550-900nucleic acids, more preferably 600-900 nucleic acids, more preferably650-900 nucleic acids, more preferably 700-900 nucleic acids, morepreferably 750-900 nucleic acids, more preferably 800-900 nucleic acids,more preferably 850-900 nucleic acids.

A preferred group of isolated polynucleotides include SEQ ID No 1 and10, which are human Meteorin cDNA sequences. Generally the cDNA sequenceis much shorter than the genomic sequences are more easily inserted intoan appropriate expression vector and transduced/fected into a productioncell or a human cell in vivo or ex vivo.

In addition, the nucleotide sequences of the invention includesequences, which are derivatives of these sequences. The invention alsoincludes vectors, liposomes and other carrier vehicles, which encompassone of these sequences or a derivative of one of these sequences. Theinvention also includes proteins transcribed and translated fromMeteorin cDNA, preferably human Meteorin cDNA, including but not limitedto human Meteorin and derivatives and variants.

Codon optimised nucleic acid molecules for enhanced expression inselected host cells, including but not limited to E. coli, yeastspecies, Chinese Hamster, Baby Hamster, insect, fungus, and human arealso contemplated.

Variant nucleic acids can be made by state of the art mutagenesismethods. Methods for shuffling coding sequences from human with those ofmouse, rat or chimpanzee are also contemplated.

Variant nucleic acids made by exchanging amino acids present in humanMeteorin with the amino acid present in mouse or rat Meteorin at thecorresponding position, should this amino acid be different from the onepresent in human Meteorin.

Viral Vectors

Broadly, gene therapy seeks to transfer new genetic material to thecells of a patient with resulting therapeutic benefit to the patient.Such benefits include treatment or prophylaxis of a broad range ofdiseases, disorders and other conditions.

Ex vivo gene therapy approaches involve modification of isolated cells(including but not limited to stem cells, neural and glial precursorcells, and foetal stem cells), which are then infused, grafted orotherwise transplanted into the patient. See, e.g., U.S. Pat. Nos.4,868,116, 5,399,346 and 5,460,959. In vivo gene therapy seeks todirectly target host patient tissue in vivo.

Viruses useful as gene transfer vectors include papovavirus, adenovirus,vaccinia virus, adeno-associated virus, herpesvirus, and retroviruses.Suitable retroviruses include the group consisting of HIV, SIV, FIV,EIAV, MoMLV. A further group of suitable retroviruses includes the groupconsisting of HIV, SIV, FIV, EAIV, CIV. Another group of preferred virusvectors includes the group consisting of alphavirus, adenovirus, adenoassociated virus, baculovirus, HSV, coronavirus, Bovine papilloma virus,Mo-MLV, preferably adeno associated virus.

Preferred viruses for treatment of disorders of the nervous system arelentiviruses and adeno-associated viruses. Both types of viruses canintegrate into the genome without cell divisions, and both types havebeen tested in pre-clinical animal studies for indications of thenervous system, in particular the central nervous system.

Methods for preparation of AAV are described in the art, e.g. U.S. Pat.No. 5,677,158. U.S. Pat. No. 6,309,634 and U.S. Pat. No. 6,683,058describe examples of delivery of AAV to the central nervous system.

Preferably, a lentivirus vector is a replication-defective lentivirusparticle. Such a lentivirus particle can be produced from a lentiviralvector comprising a 5′ lentiviral LTR, a tRNA binding site, a packagingsignal, a promoter operably linked to a polynucleotide signal encodingsaid fusion protein, an origin of second strand DNA synthesis and a 3′lentiviral LTR. Methods for preparation and in vivo administration oflentivirus to neural cells are described in US 20020037281 (Methods fortransducing neural cells using lentiviral vectors).

Retroviral vectors are the vectors most commonly used in human clinicaltrials, since they carry 7-8 kb and since they have the ability toinfect cells and have their genetic material stably integrated into thehost cell with high efficiency. See, e.g., WO 95/30761; WO 95/24929.Oncovirinae require at least one round of target cell proliferation fortransfer and integration of exogenous nucleic acid sequences into thepatient. Retroviral vectors integrate randomly into the patient'sgenome. Retroviruses can be used to target stem cells of the nervoussystem as very few cell divisions take place in other cells of thenervous system (in particular the CNS).

Three classes of retroviral particles have been described; ecotropic,which can infect murine cells efficiently, and amphotropic, which caninfect cells of many species. The third class includes xenotrophicretrovirus which can infect cells of another species than the specieswhich produced the virus. Their ability to integrate only into thegenome of dividing cells has made retroviruses attractive for markingcell lineages in developmental studies and for delivering therapeutic orsuicide genes to cancers or tumors.

For use in human patients, the retroviral vectors must be replicationdefective. This prevents further generation of infectious retroviralparticles in the target tissue—instead the replication defective vectorbecomes a “captive” transgene stable incorporated into the target cellgenome. Typically in replication defective vectors, the gag, env, andpol genes have been deleted (along with most of the rest of the viralgenome). Heterologous DNA is inserted in place of the deleted viralgenes. The heterologous genes may be under the control of the endogenousheterologous promoter, another heterologous promoter active in thetarget cell, or the retroviral 5′ LTR (the viral LTR is active indiverse tissues). Typically, retroviral vectors have a transgenecapacity of about 7-8 kb.

Replication defective retroviral vectors require provision of the viralproteins necessary for replication and assembly in trans, from, e.g.,engineered packaging cell lines. It is important that the packagingcells do not release replication competent virus and/or helper virus.This has been achieved by expressing viral proteins from RNAs lackingthe ψ signal, and expressing the gag/pol genes and the env gene fromseparate transcriptional units. In addition, in some 2. and 3.generation retriviruses, the 5′ LTR's have been replaced with non-viralpromoters controlling the expression of these genes, and the 3′ promoterhas been minimised to contain only the proximal promoter. These designsminimize the possibility of recombination leading to production ofreplication competent vectors, or helper viruses.

Expression Vectors

Construction of vectors for recombinant expression of Meteorinpolypeptides for use in the invention may be accomplished usingconventional techniques which do not require detailed explanation to oneof ordinary skill in the art. For review, however, those of ordinaryskill may wish to consult Maniatis et al., in Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, (NY 1982). Expressionvectors may be used for generating producer cells for recombinantproduction of Meteorin polypeptides for medical use, and for generatingtherapeutic cells secreting Meteorin polypeptides for naked orencapsulated therapy.

Briefly, construction of recombinant expression vectors employs standardligation techniques. For analysis to confirm correct sequences invectors constructed, the genes are sequenced using, for example, themethod of Messing, et al., (Nucleic Acids Res., 9: 309-, 1981), themethod of Maxam, et al., (Methods in Enzymology, 65: 499, 1980), orother suitable methods which will be known to those skilled in the art.

Size separation of cleaved fragments is performed using conventional gelelectrophoresis as described, for example, by Maniatis, et al.,(Molecular Cloning, pp. 133-134, 1982).

For generation of efficient expression vectors, these should containregulatory sequences necessary for expression of the encoded gene in thecorrect reading frame. Expression of a gene is controlled at thetranscription, translation or post-translation levels. Transcriptioninitiation is an early and critical event in gene expression. Thisdepends on the promoter and enhancer sequences and is influenced byspecific cellular factors that interact with these sequences. Thetranscriptional unit of many genes consists of the promoter and in somecases enhancer or regulator elements (Banerji et al., Cell 27: 299(1981); Corden et al., Science 209: 1406 (1980); and Breathnach andChambon, Ann. Rev. Biochem. 50: 349 (1981)). For retroviruses, controlelements involved in the replication of the retroviral genome reside inthe long terminal repeat (LTR) (Weiss et al., eds., The molecularbiology of tumor viruses: RNA tumor viruses, Cold Spring HarborLaboratory, (NY 1982)). Moloney murine leukemia virus (MLV) and Roussarcoma virus (RSV) LTRs contain promoter and enhancer sequences (Jollyet al., Nucleic Acids Res. 11: 1855 (1983); Capecchi et al., In:Enhancer and eukaryotic gene expression, Gulzman and Shenk, eds., pp.101-102, Cold Spring Harbor Laboratories (NY 1991). Other potentpromoters include those derived from cytomegalovirus (CMV) and otherwild-type viral promoters.

Promoter and enhancer regions of a number of non-viral promoters havealso been described (Schmidt et al., Nature 314: 285 (1985); Rossi anddeCrombrugghe, Proc. Natl. Acad. Sci. USA 84: 5590-5594 (1987)). Methodsfor maintaining and increasing expression of transgenes in quiescentcells include the use of promoters including collagen type I (1 and 2)(Prockop and Kivirikko, N. Eng. J. Med. 311: 376 (1984); Smith andNiles, Biochem. 19: 1820 (1980); de Wet et al., J. Biol. Chem., 258:14385 (1983)), SV40 and LTR promoters.

According to one embodiment of the invention, the promoter is aconstitutive promoter selected from the group consisting of: ubiquitinpromoter, CMV promoter, JeT promoter (U.S. Pat. No. 6,555,674), SV40promoter, Elongation Factor 1 alpha promoter (EF1-alpha), RSV, CAG.Examples of inducible/repressible promoters include: Tet-On, Tet-Off,Rapamycin-inducible promoter, Mx1, Mo-MLV-LTR, progesterone, RU486.

A group of preferred promoters include CAG, CMV, human UbiC, JeT, SV40,RSV, Tet-regulatable promoter, Mo-MLV-LTR, Mx1, Mt1 and EF-1alpha.

In addition to using viral and non-viral promoters to drive transgeneexpression, an enhancer sequence may be used to increase the level oftransgene expression. Enhancers can increase the transcriptionalactivity not only of their native gene but also of some foreign genes(Armelor, Proc. Natl. Acad. Sci. USA 70: 2702 (1973)). For example, inthe present invention collagen enhancer sequences may be used with thecollagen promoter 2 (I) to increase transgene expression. In addition,the enhancer element found in SV40 viruses may be used to increasetransgene expression. This enhancer sequence consists of a 72 base pairrepeat as described by Gruss et al., Proc. Natl. Acad. Sci. USA 78: 943(1981); Benoist and Chambon, Nature 290: 304 (1981), and Fromm and Berg,J. Mol. Appl. Genetics, 1: 457 (1982), all of which are incorporated byreference herein. This repeat sequence can increase the transcription ofmany different viral and cellular genes when it is present in serieswith various promoters (Moreau et al., Nucleic Acids Res. 9: 6047(1981).

Further expression enhancing sequences include but are not limited toWoodchuck hepatitis virus post-transcriptional regulation element, WPRE,SP163, CMV enhancer, and Chicken [beta]-globin insulator or otherinsulators.

Cell Lines

In one aspect the invention relates to isolated host cells geneticallymodified with the vector according to the invention.

The invention also relates to cells suitable for biodelivery of Meteorinvia naked or encapsulated cells, which are genetically modified tooverexpress Meteorin, and which can be transplanted to the patient todeliver bioactive Meteorin polypeptide locally. Such cells may broadlybe referred to as therapeutic cells.

For ex vivo gene therapy, the preferred group of cells includes neuronalcells, neuronal precursor cells, neuronal progenitor cells, neuronalstem cells, human glial stem cells, human precursor cells, stem cellsand foetal cells.

For encapsulation the preferred cells include retinal pigmentedepithelial cells, including ARPE-19 cells; human immortalisedfibroblasts; and human immortalised astrocytes.

The ARPE-19 cell line is a superior platform cell line for encapsulatedcell based delivery technology and is also useful for unencapsulatedcell based delivery technology. The ARPE-19 cell line is hardy (i.e.,the cell line is viable under stringent conditions, such as implantationin the central nervous system or the intra-ocular environment). ARPE-19cells can be genetically modified to secrete a substance of therapeuticinterest. ARPE-19 cells have a relatively long life span. ARPE-19 cellsare of human origin. Furthermore, encapsulated ARPE-19 cells have goodin vivo device viability. ARPE-19 cells can deliver an efficaciousquantity of growth factor. ARPE-19 cells elicit a negligible host immunereaction. Moreover, ARPE-19 cells are non-tumorigenic. Methods forculture and encapsulation of ARPE-19 cells are described in U.S. Pat.No. 6,361,771.

In another embodiment the therapeutic cell line is selected from thegroup consisting of: human fibroblast cell lines, human astrocyte celllines, human mesencephalic cell line, and human endothelial cell line,preferably immortalised with TERT, SV40T or vmyc.

Extracellular Matrix

The present invention further comprises culturing Meteorin producingcells in vitro on a extracellular matrix prior to implantation into themammalian nervous system. The preadhesion of cells to microcarriersprior to implantation is designed to enhance the long-term viability ofthe transplanted cells and provide long term functional benefit.

Materials of which the extracellular matrix can be comprised includethose materials to which cells adhere following in vitro incubation, andon which cells can grow, and which can be implanted into the mammalianbody without producing a toxic reaction, or an inflammatory reactionwhich would destroy the implanted cells or otherwise interfere withtheir biological or therapeutic activity. Such materials may besynthetic or natural chemical substances, or substances having abiological origin.

The matrix materials include, but are not limited to, glass and othersilicon oxides, polystyrene, polypropylene, polyethylene, polyvinylidenefluoride, polyurethane, polyalginate, polysulphone, polyvinyl alcohol,acrylonitrile polymers, polyacrylamide, polycarbonate, polypentent,nylon, amylases, natural and modified gelatin and natural and codifiedcollagen, natural and modified polysaccharides, including dextrans andcelluloses (e.g., nitrocellulose), agar, and magnetite. Eitherresorbable or non-resorbable materials may be used. Also intended areextracellular matrix materials, which are well-known in the art.Extracellular matrix materials may be obtained commercially or preparedby growing cells which secrete such a matrix, removing the secretingcells, and allowing the cells which are to be transplanted to interactwith and adhere to the matrix. The matrix material on which the cells tobe implanted grow, or with which the cells are mixed, may be anindigenous product of RPE cells. Thus, for example, the matrix materialmay be extracellular matrix or basement membrane material, which isproduced and secreted by RPE cells to be implanted.

To improve cell adhesion, survival and function, the solid matrix mayoptionally be coated on its external surface with factors known in theart to promote cell adhesion, growth or survival. Such factors includecell adhesion molecules, extracellular matrix, such as, for example,fibronectin, laminin, collagen, elastin, glycosaminoglycans, orproteoglycans or growth factors.

Alternatively, if the solid matrix to which the implanted cells areattached is constructed of porous material, the growth- or survivalpromoting factor or factors may be incorporated into the matrixmaterial, from which they would be slowly released after implantation invivo.

The configuration of the support is preferably spherical, as in a bead,but may be cylindrical, elliptical, a flat sheet or strip, a needle orpin shape, and the like. A preferred form of support matrix is a glassbead. Another preferred bead is a polystyrene bead.

Bead sizes may range from about 10 μm to 1 mm in diameter, preferablyfrom about 90 μm to about 150 μm. For a description of variousmicrocarrier beads, see, for example, isher Biotech Source 87-88, FisherScientific Co., 1987, pp. 72-75; Sigma Cell Culture Catalog, SigmaChemical Co., St, Louis, 1991, pp. 162-163; Ventrex Product Catalog,Ventrex Laboratories, 1989; these references are hereby incorporated byreference. The upper limit of the bead's size may be dictated by thebead's stimulation of undesired host reactions, which may interfere withthe function of the transplanted cells or cause damage to thesurrounding tissue. The upper limit of the bead's size may also bedictated by the method of administration. Such limitations are readilydeterminable by one of skill in the art.

Biocompatible Capsule

In one aspect the invention relates to a biocompatible capsulecontaining isolated host cells genetically modified with the vectoraccording to the invention.

Encapsulated cell biodelivery therapy is based on the concept ofisolating cells from the recipient host's immune system by surroundingthe cells with a semipermeable biocompatible material beforeimplantation within the host. The invention includes a capsule in whichcells are encapsulated in an immunoisolatory capsule. Cells areimmunoisolated from the host by enclosing them within implantablepolymeric capsules formed by a microporous membrane. This approachprevents the cell-to-cell contact between host and implanted tissues,eliminating antigen recognition through direct presentation.

The cell capsule, in the following referred to as the capsule, has amembrane which is tailored to control diffusion of molecules, such asgrowth factor hormones, neurotransmitters, peptides, antibodies andcomplements, based on their molecular weight (Lysaght et al., 56 J. CellBiochem. 196 (1996), Colton, 14 Trends Biotechnol. 158 (1996)). Usingencapsulation techniques, cells can be transplanted into a host withoutimmune rejection, either with or without use of immunosuppressive drugs.Useful biocompatible polymer capsules usually contain a core thatcontains cells, either suspended in a liquid medium or immobilisedwithin an immobilising matrix, and a surrounding or peripheral region ofpermselective matrix or membrane (“jacket”) that does not containisolated cells, that is biocompatible, and that is sufficient to protectcells in the core from detrimental immunological attack. Encapsulationhinders elements of the immune system from entering the capsule, therebyprotecting the encapsulated cells from immune destruction. Thesemipermeable nature of the capsule membrane also permits thebiologically active molecule of interest to easily diffuse from thecapsule into the surrounding host tissue and allows nutrients to diffuseeasily into the capsule and support the encapsulated cells. The capsulecan be made from a biocompatible material. A “biocompatible material” isa material that, after implantation in a host, does not elicit adetrimental host response sufficient to result in the rejection of thecapsule or to render it inoperable, for example through degradation. Thebiocompatible material is relatively impermeable to large molecules,such as components of the host's immune system, but is permeable tosmall molecules, such as insulin, growth factors, and nutrients, whileallowing metabolic waste to be removed. A variety of biocompatiblematerials are suitable for delivery of growth factors by the compositionof the invention. Numerous biocompatible materials are known, havingvarious outer surface morphologies and other mechanical and structuralcharacteristics. Preferably the capsule of this invention will besimilar to those described by WO 92/19195, WO 95/05452 or WO2005/095450, incorporated by reference; or U.S. Pat. Nos. 5,639,275;5,653,975; 4,892,538; 5,156,844; 5,283,187; or U.S. Pat. No. 5,550,050,incorporated by reference.

Such capsules allow for the passage of metabolites, nutrients andtherapeutic substances while minimizing the detrimental effects of thehost immune system. Components of the biocompatible material may includea surrounding semipermeable membrane and the internal cell-supportingscaffolding. Preferably, the recombinant cells are seeded onto thescaffolding, which is encapsulated by the permselective membrane. Thefilamentous cell-supporting scaffold may be made from any biocompatiblematerial selected from the group consisting of acrylic, polyester,polyethylene, polypropylene polyacetonitrile, polyethyleneteraphthalate, nylon, polyamides, polyurethanes, polybutester, silk,cotton, chitin, carbon, or biocompatible metals. Also, bonded fibrestructures can be used for cell implantation (U.S. Pat. No. 5,512,600).Biodegradable polymers include those comprised of poly(lactic acid) PLA,poly(lactic-coglycolic acid) PLGA, and poly(glycolic acid) PGA and theirequivalents. Foam scaffolds have been used to provide surfaces ontowhich transplanted cells may adhere (WO 2005/095450 and WO 98/05304).Woven mesh tubes have been used as vascular grafts (WO 99/52573).Additionally, the core can be composed of an immobilizing matrix formedfrom a hydrogel, which stabilizes the position of the cells. A hydrogelis a 3-dimensional network of cross-linked hydrophilic polymers in theform of a gel, substantially composed of water.

The jacket preferably has a molecular weight cutoff, defined as thatmolecular weight, where the membrane (the jacket) will reject 90% of thesolutes, of less than 1000 kD, more preferably between 50-700 kD, morepreferably between 70-300 kD, more preferably between 70-150 kD, such asbetween 70 and 130 kD. The molecular weight cutoff should be selected toensure that the bioactive molecule can escape from the capsule whileprotecting the encapsulated cells from the immune system of the patient.

The thickness of the jacket typically lies in the range of 2 to 200microns, more preferably from 50 to 150 microns. The jacket should havea thickness to give the capsule sufficient strength to keep the cellsencapsulated and should with this in mind be kept as thin as possible totake up as little space as possible.

Various polymers and polymer blends can be used to manufacture thesurrounding semipermeable membrane, including polyacrylates (includingacrylic copolymers), polyvinylidenes, polyvinyl chloride copolymers,polyurethanes, polystyrenes, polyamides, cellulose acetates, cellulosenitrates, polysulfones (including polyether sulfones), polyphosphazenes,polyacrylonitriles, poly(acrylonitrile/covinyl chloride), as well asderivatives, copolymers and mixtures thereof. Preferably, thesurrounding semipermeable membrane is a biocompatible semipermeablehollow fibre membrane. Such membranes, and methods of making them aredisclosed by U.S. Pat. Nos. 5,284,761 and 5,158,881. The surroundingsemipermeable membrane may be formed from a polyether sulfone hollowfibre, such as those described by U.S. Pat. No. 4,976,859 or U.S. Pat.No. 4,968,733. An alternate surrounding semipermeable membrane materialis poly(acrylonitrile/covinyl chloride) (Pan-PVC).

The capsule can be any configuration appropriate for maintainingbiological activity and providing access for delivery of the product orfunction, including for example, cylindrical, rectangular, disk-shaped,patch-shaped, ovoid, stellate, or spherical. Moreover, the capsule canbe coiled or wrapped into a mesh-like or nested structure. If thecapsule is to be retrieved after it is implanted, configurations, whichtend to lead to migration of the capsules from the site of implantation,such as spherical capsules small enough to travel in the recipienthost's blood vessels, are not preferred. Certain shapes, such asrectangles, patches, disks, cylinders, and flat sheets offer greaterstructural integrity and are preferable where retrieval is desired. Aparticularly preferred shape is cylinder-shaped as such a shape iseasily produced from hollow fibres which can be produced industrially.

A macrocapsule in the present context is a capsule having a volume of atleast 1 μL, such as from 1 to 10 μL.

When macrocapsules are used, preferably at least 10³ cells areencapsulated, such as between 10³ and 10⁸ cells are encapsulated, mostpreferably 10⁵ to 10⁷ cells are encapsulated in each device. Of course,the number of cells in each capsule depends on the size of the capsule.As a rule of thumb, in a capsule with foam (described below) the presentinventors have found that loading between 10,000 and 100,000 cells perμL of capsule (volume calculated as the internal volume including foam)results in a good filling of the capsule, more preferably from 25,000 to50,000 cells per μL, more preferably from 30,000 to 40,000 cells per μL.The number of cells to be loaded also depends on the size of the cells.

Dosage may be controlled by varying the dimensions (length, diameter) ofthe capsule and/or by implanting a fewer or greater number of capsules,preferably between 1 and 10 capsules per patient.

The scaffolding may be coated with extracellular matrix (ECM) molecules.Suitable examples of extracellular matrix molecules include, forexample, collagen, laminin, and fibronectin. The surface of thescaffolding may also be modified by treating with plasma irradiation toimpart charge to enhance adhesion of cells.

Any suitable method of sealing the capsules may be used, including theuse of polymer adhesives or crimping, knotting and heat sealing. Inaddition, any suitable “dry” sealing method can also be used, asdescribed, e.g., in U.S. Pat. No. 5,653,687.

The encapsulated cell devices are implanted according to knowntechniques. Many implantation sites are contemplated for the devices andmethods of this invention. These implantation sites include, but are notlimited to, the central nervous system, including the brain, spinal cord(see, U.S. Pat. Nos. 5,106,627, 5,156,844, and 5,554,148), and theaqueous and vitreous humors of the eye (see WO 97/34586).

The disclosed capsule may include an integral tether that extends fromthe capsule and which is of a length sufficient to reach at least fromthe treatment site to the proximity of the insertion site therebyfacilitating fixation of the capsule at the insertion site, e.g. to theouter surface of the skull. The insertion site is subsequently coveredby skin.

To facilitate removal of the capsule from the tissue, e.g. when thetreatment comes to an end, or if the capsule must be replaced, thetransition between the capsule and the tether could be smooth andwithout projections of any kind, or the dimension could be increasedfrom the capsule towards the tether. This, obviously, creates an edgebetween the two parts but since the relatively small capsule forms thedistal end of the therapy system, i.e. the end which is towards thebody, ancillary damage may be prevented during removal of the capsule.If the capsule and the tether are tubular with circular cross sectionalshapes, the radial size of the capsule may therefore preferably besmaller than the radial size of the tether, and the capsule and tethermay preferably be joined coaxially to each other. Preferably the capsuleof this invention will be similar in design to those described by WO2006/122551 and WO 2005/095450.

Capsules may be filled by using a syringe or alternatively, automated orsemi-automated filling may be used as described in WO2007/048413.

EXAMPLES Example 1 Protein Purification

Mouse meteorin (Uniprot Accession # Q8C1Q4; SEQ ID NO:5) (aa22-291 (SEQID NO:6) with a signal peptide from hCD33) was cloned into an expressionvector. The vector was transfected into the NS0 mouse myeloma cell lineby electroporation.

Stable clones were isolated and screened for expression of mMeteorin byWestern analysis using Gt×mMETRN polyclonal antibody (AF3475).Conditioned medium from cultures containing mouse Meteorin wasconcentrated, supplemented with 20 mM MOPS, the pH was adjusted to 6.5,and filtered through a 0.2 um filter. The sample was applied to an anionexchange chromatography resin, equilibrated in 20 mM MOPS, 0.1 M NaCl,pH 6.5. The fractions containing mouse Meteorin were supplemented with 2M NaCl, the pH was adjusted to 7.0, and then applied to a phenylsepharose resin. Bound proteins were eluted with a decreasing gradientof NaCl. Fractions enriched in mouse Meteorin were pooled, concentratedand loaded onto a Superdex gel filtration column and then equilibratedin PBS. Mouse Meteorin eluted as an approximately 30 kDa molecularweight protein. Fractions of interest were pooled, concentrated,dialyzed against PBS and stored at −80 C.

Example 2 Photochemically Induced Sciatic Nerve Injury

The effect of systemically (sc) administered Meteorin was investigatedin photochemically induced sciatic nerve injured rats known to developallodynia to both mechanical and cold stimulation within one week afterinjury (Kupers, R., Yu, W., Persson, J. K., Xu, X. J., andWiesenfeld-Hallin, Z. (1998); Photochemically-induced ischemia of therat sciatic nerve produces a dose-dependent and highly reproduciblemechanical, heat and cold allodynia, and signs of spontaneous pain. Pain76, 45-59). Briefly, after unilateral photoinduced injury of the sciaticnerve, animals were randomly divided into four groups (n=8 per group)and injected with saline as negative control or Meteorin at threedifferent concentrations (0.05, 0.2 and 0.8 mg/kg). Each rat receivedsix injections over a two week period starting after seven days when astable allodynia was developed. Behavioral assessments were conductedbefore each injection during the treatment period and for two additionalweeks.

From FIG. 1 it is evident that saline and Meteorin at the low dose (0.05mg/kg) did not affect the response to mechanical stimulation of theipsilateral hind paw. 0.2 mg/kg Meteorin reduced mechanical allodyniamoderately but this group was not statistically different from thesaline control group. In contrast, repeated injection of 0.8 mg/kgMeteorin produced a significant and marked alleviation of mechanicalallodynia. After treatment cessation on Day 21, this group stayedsignificantly different from vehicle for at least a week. Over time,mechanical allodynia was gradually reestablished.

The response to cold was evaluated by briefly spraying ethyl chloride onthe plantar surface of the hind paw and scoring animal behavioraccordingly. FIG. 2 shows that treatment with 0.8 mg/kg Meteorinpotently alleviated cold allodynia stimulation and that the 0.2 mg/kgalso has a significant positive effect. After treatment cessation, thegroup treated with 0.8 mg/kg Meteorin stayed significantly differentfrom vehicle for at least a week and there was a trend towardsimprovement even after two weeks. The cold allodynia graduallyreestablished over time but this was not complete at the end of theexperiment. There was no effect of 0.05 mg/kg Meteorin which was similarto the control group thoughout the study.

Importantly, all animals gained weight normally throughout the study andno side effects were observed (FIG. 3).

In conclusion, Meteorin dose-dependently reduced both mechanical andcold allodynia and the effect lasted for at least a week after treatmentcessation. Two weeks after treatment cessation, hypersensivity seemed togradually reestablish. No side effects were observed.

Example 3 Chronic Constriction Injury (CCI)

The effect of Meteorin was further investigated in the well establishedchronic constriction injury (CCI) model (Bennett, G. J., and Xie, Y. K.(1988); A peripheral mononeuropathy in rat that produces disorders ofpain sensation like those seen in man. Pain 33, 87-107). Briefly, twelvedays after injury, when a stable mechanical allodynia was established,animals received six subcutaneous injections of Meteorin (0.1, 0.5, 2.0mg/kg) or vehicle distributed over the next two weeks (n=7-8 per group).Animal behavior was followed throughout the study up to three weeksafter the last injection.

Weight bearing was evaluated immediately before and after treatment as asurrogate maker for spontaneous pain. Prior to treatment (Day 12), allgroups had a side-to-side deficit of approximately 50 g which wasreduced to 10-15 g for the groups treated with Meteorin. After treatmentcessation, the effect was gradually reduced and there was no significantdifference between the groups after three weeks.

Mechanical allodynia was also evaluated in the CCI animals. The averagebaseline paw withdrawal threshold to mechanical stimulation withcalibrated von Frey hairs was 15 g which was gradually reduced to 2 g onDay 12 where treatment began. While the vehicle group remainedhypersensitive throughout the study (−2 g), Meteorin effectivelyalleviated mechanical allodynia at all doses tested (8-11 g). Aftertreatment cessation, animals in the Meteorin treatment group remainedsignificantly different from the control group for approximately a weekbut allodynia was gradually reestablished and there was no differencebetween groups after three weeks. To evaluate the magnitude of theeffect, animals in the control group were given a high dose ofGabapentin (200 mg/kg) for comparison. One hour after Gabapentintreatment the threshold to mechanical stimulation was 9.7±1.9 g comparedto 1.9±0.7 g before treatment. It is clear that Meteorin and Gabapentinare similarly effective but importantly while Gabapentin is ananalgesic, Meteorin has long lasting and potentially disease modifyingeffects.

Meteorin injections did not cause weight loss or general behavioraldifferences between control and treated animals.

Example 4 Chronic Constriction Injury (CCI) Objective

This study was designed to investigate the efficacy of sub cutaneously(s.c) administered recombinant Meteorin to alleviating allodynia andspontaneous pain in rats produced by chronic constriction injury (CCI)(Bennett and Xie, “A peripheral mononeuropathy in rat that producesdisorders of pain sensation like those seen in man”, 1988, Pain 33; p.87-107).

Methods

Recombinant Meteorin. Recombinant mouse Meteorin (Uniprot Accession #Q8C1Q4) was produced as described elsewhere in this application.

Surgery: 30 male Sprague-Dawley rats weighing 250-280 g underwentsurgery to produce a chronic constriction of the left sciatic nerveusing four loose ligatures of 4-0 chromic gut suture (CCI Model)(Bennett and Xie, “A peripheral mononeuropathy in rat that producesdisorders of pain sensation like those seen in man”, 1988, Pain 33; p.87-107). Rats were anesthetized via inhalation of isofluorane gas. Ratsreceived a skin incision just caudal to the biceps femoris at mid-thighlevel on the left hindlimb. A small incision was then made into theunderlying muscle layer and separated gently using hemostats with caretaken not to disturb the sciatic nerve. The sciatic nerve was thenidentified, freed of adhering tissue and slightly elevated using 45°angle forceps. Four pieces of 4-0 chromic gut suture material(previously washed in sterile saline) were brought under the nerve andthen each loosely tied around the nerve into a square knot. The knotswere spaced 1 mm apart. These loose ligatures allowed for a chronicconstriction of the nerve without cutting off blood supply. Musclelayers were sutured closed with 4-0 vicryl suture and skin closed withwound clips.

Grouping and Behavioral Analysis: At day 0 of the experiment, all ratswere tested for mechanical allodynia using Von Frey Filaments, thermalallodynia using the Hargreaves' method and weight bearing on hind limbsusing an incapacitance meter. 24 rats were selected to continue in thestudy and later divided into four treatment groups (n=6). Animals wereinjected five times s.c. with either vehicle, 0.1, 0.5 or 1.8 mg/kg ofMeteorin protein on post surgical days 10, 12, 14, 17 and 19. Animalswere further tested for mechanical and thermal allodynia as well asincapacitance at days 10, 12, 14, 17, 19, 21, 26, 32 and 39 postsurgery. Importantly, behavioral analysis was done prior to injection ofMeteorin in order to exclude immediate analgesic effects and to focus onlong lasting potentially disease modifying effects. Animals wereobserved and body weight followed throughout the study. The experimenterwas blind to treatment condition and no animals were removed from thestudy.

Assay for Meteorin in serum: Following behavioral testing onpost-surgical day 39, animals were dosed at 0.1, 0.5 and 2 mg/kg ofMeteorin and rat serum samples were collected at 2, 6, 24 hr followingdrug administration. Serum was also collected from non-treated controlrats. There were two rats for each time point. All rat serum sampleswere assayed by mouse Meteorin ELISA (R&D Systems, DY3475).

Results

Experimental allodynia and spontaneous pain were induced in rats by CCI(Bennett and Xie, 1988) and tactile allodynia evaluated using Von FreyHairs (FIG. 7). Rats had a baseline withdrawal threshold ofapproximately 15 g which was reduced to 1.5 g 10 days after the CCI. Itis evident that treatment with Meteorin rapidly reduced the allodyniaand the force withstood by 1.8 mg/kg of Meteorin treated rats weresignificant at day 17, 19, 21, 26 and 32 compared to vehicle treatedrats. As such, the significant difference was maintained at least 13days after treatment cessation and a trend towards reduced allodynia wasalso observed after 20 days. Most animals in the group treated with 1.8mg/kg of Meteorin reverted to 15 g but one animal did not respond whichexplains the increased standard error in this particular group.

With respect to thermal sensivity (FIG. 8), rats had a baselinewithdrawal latency of 16.5 seconds which was reduced to approximately 7seconds 10 days after CCI signifying a thermal allodynia. Vehicletreated animals stayed hypersensitive throughout the study whiletreatment with Meteorin at 1.8 mg/kg rapidly resulted in a significantdecrease in paw withdrawal latency from day 14 which lasted for the restof the experiment including at least three weeks after treatmentcessation. Interestingly, instead of going back to the allodynia levelof the vehicle group, the paw withdrawal latency levelled out at 10.5seconds for this Meteorin group. The 0.5 mg/kg dose of Meteorin alsoresulted in decreased paw withdrawal latency becoming significant atdays 19 and 21. There was also a trend towards decreased allodynia with0.1 mg/kg Meteorin although this did not reach statistically significantlevels. In summary, Meteorin dose-dependently reduced thermal allodyniawith significant effects in the treatment period and beyond.

At the post-surgical baseline screen, rats distributed equal weightbetween both of their hindlimbs (FIG. 9). However, following the CCIinjury there was approximately 60% less weight applied to theipsilateral hindlimb which is taken as a surrogate marker forspontaneous pain. The weight bearing deficit of 60% was maintained inthe vehicle group throughout the study. In contrast, both 0.5 and 1.8mg/kg Meteorin quickly reduced the weight bearing deficit and in bothcases did the positive effect remain significantly improved for at leastthree weeks after treatment cessation. A statistically significanteffect was also seen with the low dose Meteorin at day 19. Generally,from day 26 to the end of the experiment, the weight bearing deficitsettled in all Meteorin treated groups at steady levels lower than thevehicle group. Where the vehicle control group remained above 60%, theaverage weight bearing deficits for the Meteorin treated groups settledaround 55%, 48% and 40% respectively for 0.1, 0.5 and 1.8 mg/kgMeteorin.

No immediate side effects were observed and all animals gained weightnormally throughout the study (FIG. 10).

Following the last behavioral test on Day 39, animals were dosed with0.1, 0.5 and 1.8 mg/kg Meteorin and serum samples collected 2, 6 and 24hours later for pharmacokinetic evaluation (FIG. 11). Meteorin wasbarely detectable in serum after injection of 0.1 mg/kg but a goodrelationship between dose and serum concentration was observed betweenthe two higher doses. It is furthermore clear from FIG. 11 that Meteorinis no longer detectable in serum 24 hours after injection. In relationto this, it is interesting that the observed beneficial effects last forseveral weeks after the last injection where Meteorin is no longerpresent in serum (FIGS. 7, 8 and 9). Also, instead of returning to thehypersensitive base line level, Meteorin treatment leads to a new lesshypersensitive level. Taken all together, it is likely that Meteorin hasdisease modifying properties. As such, the long lasting effect mayreflect normalization or restoration of neuronal function.

Conclusion

Administration of Meteorin to animals that were surgically prepared toexhibit a allodynia and spontaneous pain-like syndrome resulted inprofound reduction of allodynia and spontaneous pain inferred by areduction in both thermal and tactile allodynia and a normalization ofdifferential weight bearing. Even though Meteorin is absent from serum24 hours after injections, the positive effects last for several weeksthereby demonstrating disease modifying properties.

Example 5 Photochemically Induced Sciatic Nerve Injury, IntrathecalAdministration Methods

Surgery. Male Sprague-Dawley rats (Harlan, The Netherlands) weighing380-450 g were fitted with a chronic intrathecal catheter with the tipat the lumbar enlargement (Storkson, R. V., Kjorsvik, A., Tjolsen, A.,and Hole, K. (1996). Lumbar catheterization of the spinal subarachnoidspace in the rat. J. Neurosci. Methods 65, 167-172). Three to five daysafter cathether implantation, ischemic sciatic nerve injury was producedusing a photochemical method (Kupers, R., Yu, W., Persson, J. K., Xu, X.J., and Wiesenfeld-Hallin, Z. (1998); Pain 76, 45-59). Briefly, undergeneral anesthesia (chloral hydrate 300 mg/kg), the left sciatic nervewas exposed at mid-thigh level and irradiated for 1.5 min with an argonlaser operating at 514 nm at an average power of 0.17 W. Erythrosin B(32.5 mg/kg dissolved in 0.9% saline) was injected intravenously throughthe tail vein just prior to irradiation. This operation leads to ahighly reproducible hypersensitivity within 7 days.

Evaluation of allodynia. For evaluation of mechanical allodynia, a setof calibrated nylon monofilaments (von Frey hairs, Stoelting, IL) wasapplied to the glabrous skin of the paws with increasing force until theanimal withdraws the limb. Each monofilament was applied 5 times andwithdrawal threshold was determined as the force at which the animalwithdraws the paw from at least 3 out of 5 consecutive stimuli. Theresponse to cold was tested with ethyl chloride, which was briefly (<1s) sprayed on the plantar surface of the hind paw. The response wasscored as the following: 0=no response, 1=startle-like response, nohindpaw withdrawal (normal), 2=brief withdrawal of the stimulatedhindpaw (mild pain), 3=sustained or repeated withdrawal of thestimulated hindpaw, brief licking or shaking (severe pain). All testswere performed by an experimenter who was blind with respect to theexperimental conditions.

Experimental setup. Baseline responses were evaluated after catheterimplantation and again before sciatic nerve irradiation. Rats thatdeveloped allodynia to mechanical and cold stimulation 7 days afternerve injury were randomly divided into four groups (N=8) which weregiven vehicle as negative control and three doses of recombinantMeteorin (0.5, 2 and 6 ug) at a volume of 10 μl intrathecally. Each ratreceived six injections over a two week period (on day 7, 9, 11, 14, 16and 18 counting from the time of nerve injury). Behavioral testing wasconducted prior to intrathecal injection on respective treatment daysand furthermore on days 21, 25, 28 and 35 following treatment cessation.

Results

As seen in FIG. 12, the baseline paw withdrawal threshold to mechanicalstimulation was about 50 g. 7 days after photochemically induced sciaticnerve injury, rats developed significant mechanical allodynia evident asa reduced paw withdrawal threshold of approximately 8 g. Rats were thenrandomly divided into four groups subsequently receiving either vehicleor Meteorin as six intrathecal injections in the space of two weeks.With respect to Meteorin, rats received 0.5 μg, 2 μg or 6 μg. It isclear that intrathecal injection of Meteorin significantly anddose-dependently reduced mechanical allodynia (FIG. 12). The mechanicalallodynia was gradually reestablished within a week after treatmentcessation. Intrathecal injection of vehicle did not affect themechanical hypersensitivity throughout the experiment.

As seen in FIG. 13, the baseline cold response is 1 corresponding to anormal startle-like response. 7 days after photochemically inducedsciatic nerve injury, rats developed a marked cold allodynia evident asa mild pain reaction. Treatment with 2 μg and 6 μg Meteorin quicklyreversed the cold allodynia and animals had a near normal response tocold in the treatment period. A significant positive effect of 6 μgMeteorin was also observed three days after treatment cessation.However, cold allodynia was fully reestablished a week after thetreatment ended. Vehicle had no effect on cold allodynia.

Conclusion

Repeated intrathecal injection of Meteorin significantly reducesmechanical and cold allodynia in rats after ischemic sciatic nerveinjury.

Example 6 Sequence Listing

SEQ ID NO 1: human Meteorin cDNASEQ ID NO 2: human Meteorin full length amino acid sequenceSEQ ID NO 3: human Meteorin amino acid sequence without signal peptideSEQ ID NO 4: mouse Meteorin cDNASEQ ID NO 5: mouse Meteorin full length amino acid sequenceSEQ ID NO 6: mouse Meteorin amino acid sequence without signal peptideSEQ ID NO 7: rat Meteorin cDNASEQ ID NO 8: rat Meteorin full length amino acid sequenceSEQ ID NO 9: rat Meteorin amino acid sequence without signal peptideSEQ ID NO 10: human codon optimized DNA sequenceSEQ ID NO 11: mature Meteorin, consensus sequence

Human Meteorin cDNA (1109 bp; CDS =118-999) >gi|34147349|ref|NM_024042.2| Homo sapiens hypothetical proteinMGC2601 (MGC2601), mRNA (SEQ ID NO 1)GCTTCGCCGGGGCCGGGCGGCCGGCGCCCCCGGCTGCTCCCGCCGCCGCCCGGACCCGCGCCCCGCCGGGGCAGCGGTGGTGAGAGCCCCGACTCCCCGGACGCCGCCCGCCGTGCCATGGGGTTCCCGGCCGCGGCGCTGCTCTGCGCGCTGTGCTGCGGCCTCCTGGCCCCGGCTGCCCGCGCCGGCTACTCCGAGGAGCGCTGCAGCTGGAGGGGCAGCGGCCTCACCCAGGAGCCCGGCAGCGTGGGGCAGCTGGCCCTGGCCTGTGCGGAGGGCGCGGTTGAGTGGCTGTACCCGGCTGGGGCGCTGCGCCTGACCCTGGGCGGCCCCGATCCCAGAGCGCGGCCCGGCATCGCCTGTCTGCGGCCGGTGCGGCCCTTCGCGGGCGCCCAGGTCTTCGCGGAGCGCGCAGGGGGCGCCCTGGAGCTGCTGCTGGCCGAGGGCCCGGGCCCGGCAGGGGGCCGCTGCGTGCGCTGGGGTCCCCGCGAGCGCCGGGCCCTCTTCCTGCAGGCCACGCCGCACCAGGACATCAGCCGCCGCGTGGCCGCCTTCCGCTTTGAGCTGCGCGAGGACGGGCGCCCCGAGCTGCCCCCGCAGGCCCACGGTCTCGGCGTAGACGGTGCCTGCAGGCCCTGCAGCGACGCTGAGCTGCTCCTGGCCGCATGCACCAGCGACTTCGTAATTCACGGGATCATCCATGGGGTCACCCATGACGTGGAGCTGCAGGAGTCTGTCATCACTGTGGTGGCCGCCCGTGTCCTCCGCCAGACACCGCCGCTGTTCCAGGCGGGGCGATCCGGGGACCAGGGGCTGACCTCCATTCGTACCCCACTGCGCTGTGGCGTCCACCCGGGCCCAGGCACCTTCCTCTTCATGGGCTGGAGCCGCTTTGGGGAGGCCCGGCTGGGCTGTGCCCCACGATTCCAGGAGTTCCGCCGTGCCTACGAGGCTGCCCGTGCTGCCCACCTCCACCCCTGCGAGGTGGCGCTGCACTGAGGGGCTGGGTGCTGGGGAGGGGCTGGTAGGAGGGAGGGTGGGCCCACTGCTTTGGAGGTGATGGGACTATCAATAAGAACTCTGTTCACGCAAAAAAAAAAAAAAAAAAAHuman Meteorin full length amino acid sequence >IPI00031531.1 REFSEQ_NP:NP_076947 TREMBL:Q9UJH9ENSEMBL:ENSP00000219542 Tax_Id = 9606 C380A1.2.1 (Novel protein)(SEQ ID NO 2)MGFPAAALLC ALCCGLLAPA ARAGYSEERC SWRGSGLTQE PGSVGQLALA CAEGAVEWLYPAGALRLTLG GPDPRARPGI ACLRPVRPFA GAQVFAERAG GALELLLAEG PGPAGGRCVRWGPRERRALF LQATPHQDIS RRVAAFRFEL REDGRPELPP QAHGLGVDGA CRPCSDAELLLAACTSDFVI HGIIHGVTHD VELQESVITV VAARVLRQTP PLFQAGRSGD QGLTSIRTPLRCGVHPGPGT FLFMGWSRFG EARLGCAPRF QEFRRAYEAA RAAHLHPCEV ALHHuman Meteorin, protein without signal peptide (SEQ ID NO 3)GYSEERCSWR GSGLTQEPGS VGQLALACAE GAVEWLYPAG ALRLTLGGPD PRARPGIACLRPVRPFAGAQ VFAERAGGAL ELLLAEGPGP AGGRCVRWGP RERRALFLQA TPHQDISRRVAAFRFELRED GRPELPPQAH GLGVDGACRP CSDAELLLAA CTSDFVIHGI IHGVTHDVELQESVITVVAA RVLRQTPPLF QAGRSGDQGL TSIRTPLRCG VHPGPGTFLF MGWSRFGEARLGCAPRFQEF RRAYEAARAA HLHPCEVALHMouse Meteorin cDNA, 1363 bp, CDS 84..959NM_133719. Mus musculus meteorin.[gi: 56550040] (SEQ ID NO 4)gggcagccgc gccgcgggct gctcgcgctg cggccccgac cctcccgggg cagcagtccgaggccccggc gcgtccccta accatgctgg tagccacgct tctttgcgcg ctctgttgcggcctcctggc cgcgtccgct cacgctggct actcggaaga ccgctgcagc tggaggggcagcggtttgac ccaggagcct ggcagcgtgg ggcagctgac cctggactgt actgagggcgctatcgagtg gctgtaccca gctggggcgc tgcgcctgac cctgggcggc cccgatccgggcacacggcc cagcatcgtc tgtctgcgcc cagagcggcc cttcgctggt gcccaggtcttcgctgaacg tatgaccggc aatctagagt tgctactggc cgagggcccg gacctggctgggggccgctg catgcgctgg ggtccccgcg agcgccgagc ccttttcctg caggccacaccacaccgcga catcagccgc agagttgctg ccttccgttt tgaactgcac gaggaccaacgtgcagaaat gtctccccag gctcaaggtc ttggtgtgga tggtgcctgc aggccctgcagtgatgccga gctcctcctg gctgcatgca ccagtgattt tgtgatccac gggaccatccatggggtcgc ccatgacaca gagctgcaag aatcagtcat cactgtggtg gttgctcgtgtcatccgcca gacactgcca ctgttcaagg aagggagctc ggagggccaa ggccgggcctccattcgtac cttgctgcgc tgtggtgtgc gtcctggccc aggctccttc ctcttcatgggctggagccg atttggcgaa gcttggctgg gctgtgctcc ccgcttccaa gagttcagccgtgtctattc agctgctctc acgacccatc tcaacccatg tgagatggca ctggactgagagacctggga gcaagccctg gatggacctt cttctggaga tggggtgttg gggagggtgatgggagggtg ggtgagaagg gtgtggctcg gatggcatcc tggtacccac agtgagctggtagaatacta agtaatctgg accataccag ccactgtagt catggtcttc tgtggcaggcagcataccca gctctgtgcc tgcctcactt tgtctactct ccagtctgct gcccttctaacccttcttag cctgctgacc agtgagctca tgttttcctc gaattccagg gtgctgctggggttcagagc aaccgtgccg tagtttggaa gacttgagct aattgttttt tttttgtttgtttttttgtt tgtttaaagg tggcctgggg ggggcggcaa acaMouse Meteorin full length amino acid sequence ref|NP_598480.1|meteorin [Mus musculus] (SEQ ID NO 5)MLVATLLCAL CCGLLAASAH AGYSEDRCSW RGSGLTQEPG SVGQLTLDCT EGAIEWLYPAGALRLTLGGP DPGTRPSIVC LRPERPFAGA QVFAERMTGN LELLLAEGPD LAGGRCMRWGPRERRALFLQ ATPHRDISRR VAAFRFELHE DQRAEMSPQA QGLGVDGACR PCSDAELLLAACTSDFVIHG TIHGVAHDTE LQESVITVVV ARVIRQTLPL FKEGSSEGQG RASIRTLLRCGVRPGPGSFL FMGWSRFGEA WLGCAPRFQE FSRVYSAALT THLNPCEMAL DMouse Meteorin protein without signal peptide (SEQ ID NO 6)GYSEDRCSWR GSGLTQEPGS VGQLTLDCTE GAIEWLYPAG ALRLTLGGPD PGTRPSIVCL RPERPFAGAQVFAERMTGNL ELLLAEGPDL AGGRCMRWGP RERRALFLQA TPHRDISRRV AAFRFELHED QRAEMSPQAQGLGVDGACRP CSDAELLLAA CTSDFVIHGT IHGVAHDTEL QESVITVVVA RVIRQTLPLF KEGSSEGQGRASIRTLLRCG VRPGPGSFLF MGWSRFGEAW LGCAPRFQEF SRVYSAALTT HLNPCEMALDRat Meteorin cDNA (1026 bp; CDS = 1-876) >gi|34870570|ref|XM_213261.2|Rattus norvegicus similar to 1810034Bl6Rik protein (LOC287151), mRNA(SEQ ID NO 7)ATGCTGGTAGCGGCGCTTCTCTGCGCGCTGTGCTGCGGCCTCTTGGCTGCGTCCGCTCGAGCTGGCTACTCCGAGGACCGCTGCAGCTGGAGGGGCAGCGGTTTGACCCAGGAACCTGGCAGCGTGGGGCAGCTGACCCTGGATTGTACTGAGGGTGCTATCGAGTGGCTGTATCCAGCTGGGGCGCTGCGCCTGACTCTAGGCGGCTCTGATCCGGGCACGCGGCCCAGCATCGTCTGTCTGCGCCCAACACGGCCCTTCGCTGGTGCCCAGGTCTTCGCTGAACGGATGGCCGGCAACCTAGAGTTGCTACTGGCCGAGGGCCAAGGCCTGGCTGGGGGCCGCTGCATGCGCTGGGGTCCTCGCGAGCGCCGAGCCCTTTTCCTGCAGGCCACGCCACACCGGGACATCAGCCGCAGAGTTGCTGCCTTCCAATTTGAACTGCACGAGGACCAACGTGCAGAAATGTCTCCCCAGGCCCAAGGTTTTGGTGTGGATGGTGCCTGCAGGCCCTGCAGTGATGCCGAGCTCCTTCTGACTGCATGCACCAGTGACTTTGTGATCCATGGGACCATCCATGGGGTCGTCCATGACATGGAGCTGCAAGAATCAGTCATCACTGTGGTGGCCACTCGTGTCATCCGCCAGACACTGCCACTGTTCCAGGAAGGGAGCTCGGAGGGCCGGGGCCAGGCCTCCGTTCGTACCTTGTTGCGCTGTGGTGTGCGTCCTGGCCCAGGCTCCTTCCTCTTCATGGGCTGGAGCCGATTTGGCGAAGCTTGGCTGGGCTGCGCTCCCCGCTTCCAAGAGTTCAGCCGTGTCTATTCAGCTGCTCTCGCGGCCCACCTCAACCCATGTGAGGTGGCACTGGACTGAGAGACCTGGGAGCAAGCCCTGGATGGATCTTCCTCTGGGGATGGGGTGTTGGGGAGGGGTGATAGGAGGGTGGGTGGGAAGGGTGTGGCTCAGATGGCATCCTGGTACCCACAGTGAGGTGGTAGAATACTAAATAACCTGGATCACACCRat Meteorin full length amino acid sequence >IPI00369281.1 |REFSEQ_XP:XP_213261|ENSEMBL:ENSRNOP00000026676(SEQ ID NO 8)MLVAALLCAL CCGLLAASAR AGYSEDRCSW RGSGLTQEPG SVGQLTLDCT EGAIEWLYPAGALRLTLGGS DPGTRPSIVC LRPTRPFAGA QVFAERMAGN LELLLAEGQG LAGGRCMRWGPRERRALFLQ ATPHRDISRR VAAFQFELHE DQRAEMSPQA QGFGVDGACR PCSDAELLLTACTSDFVIHG TIHGVVHDME LQESVITVVA TRVIRQTLPL FQEGSSEGRG QASVRTLLRCGVRPGPGSFL FMGWSRFGEA WLGCAPRFQE FSRVYSAALA AHLNPCEVAL DRat Meteorin, protein without signal peptide (SEQ ID NO 9)GYSEDRCSWR GSGLTQEPGS VGQLTLDCTE GAIEWLYPAG ALRLTLGGSD PGTRPSIVCLRPTRPFAGAQ VFAERMAGNL ELLLAEGQGL AGGRCMRWGP RERRALFLQA TPHRDISRRVAAFQFELHED QRAEMSPQAQ GFGVDGACRP CSDAELLLTA CTSDFVIHGT IHGVVHDMELQESVITVVAT RVIRQTLPLF QEGSSEGRGQ ASVRTLLRCG VRPGPGSFLF MGWSRFGEAWLGCAPRFQEF SRVYSAALAA HLNPCEVALDCodon optimized Meteorin nucleotide sequence present in constructspCAn.Meteorin and pT2.CAn.Meteorin (SEQ ID NO 10)ATGGGCTTTCCCGCTGCCGCCCTGCTGTGCGCTCTGTGCTGCGGACTGCTGGCTCCTGCAGCCAGAGCCGGCTACAGCGAGGAACGGTGCAGCTGGCGGGGCAGCGGCCTGACCCAGGAACCTGGCAGCGTCGGCCAGCTCGCACTGGCCTGTGCAGAAGGCGCCGTGGAGTGGCTGTACCCCGCAGGCGCCCTGAGACTGACCCTGGGCGGACCCGACCCCAGAGCCAGACCCGGCATTGCCTGTCTGAGGCCCGTGCGGCCTTTCGCTGGCGCCCAGGTGTTCGCCGAGAGAGCCGGCGGAGCCCTGGAACTCCTGCTCGCCGAAGGCCCTGGTCCAGCCGGCGGAAGATGCGTGAGATGGGGCCCAAGAGAGCGGAGAGCCCTGTTCCTGCAAGCCACCCCCCACCAGGACATCAGCAGACGGGTGGCCGCCTTCAGATTCGAGCTGCGGGAGGACGGTAGACCCGAGCTGCCACCTCAGGCCCACGGACTGGGAGTGGACGGCGCCTGCAGACCCTGTAGCGACGCCGAGCTGCTGCTCGCCGCCTGCACCAGCGACTTCGTGATCCACGGCATCATCCACGGCGTGACCCACGACGTGGAGCTGCAGGAAAGCGTCATCACCGTCGTCGCCGCCAGAGTGCTGAGACAGACCCCCCCTCTGTTCCAGGCCGGCAGAAGCGGCGACCAGGGCCTGACCAGCATCCGGACCCCCCTGAGATGCGGCGTGCATCCCGGACCCGGCACCTTCCTGTTCATGGGCTGGTCCAGATTCGGCGAGGCCCGGCTGGGCTGCGCTCCCCGGTTCCAGGAATTCAGACGGGCCTACGAGGCCGCCAGGGCCGCTCATCTGCACCCCTGCGAGGTGGCCCTGCATTGA Consensus sequence, mature Meteorin(SEQ ID NO 11)GYSEXRCSWR GSGLTQEPGS VGQLXLXCXE GAXEWLYPAG ALRLTLGGXD PXXRPXIXCL  60RPXRPFAGAQ VFAERXXGXL ELLLAEGXXX AGGRCXRWGP RERRALFLQA TPHXDISRRV 120AAFXFELXED XRXEXXPQAX GXGVDGACRP CSDAELLLXA CTSDFVIHGX IHGVXHDXEL 180QESVITVVXX RVXRQTXPLF XXGXSXXXGX XSXRTXLRCG VXPGPGXFLF MGWSRFGEAX 240LGCAPRFQEF XRXYXAAXXX HLXPCEXALX                                  270 Xis any of the 21 amino acids that can be encoded by DNA.

1. A method of treatment of allodynia in a subject comprisingadministering to said subject in need thereof therapeutically effectiveamounts of a neurotrophic polypeptide comprising an amino acid sequencehaving at least 85% sequence identity to SEQ ID NO:
 3. 2. The method ofclaim 1, wherein said polypeptide has at least 90% sequence identity toSEQ ID NO:
 3. 3. The method of claim 1, wherein the neurotrophicpolypeptide comprises the consensus sequence of SEQ ID NO:11.
 4. Themethod of claim 1, wherein the neurotrophic polypeptide has cysteineresidues at positions 7, 28, 59, 95, 148, 151, 161, 219, 243, and 265relative to the amino acid sequence of SEQ ID NO:3.
 5. The method ofclaim 1, wherein the neurotrophic polypeptide is a variant polypeptidedescribed therein, wherein any amino acid substitutions are conservativesubstitutions.
 6. The method of claim 1, wherein said polypeptide iscapable of forming at least one intramolecular cysteine bridge.
 7. Themethod according to claim 1, wherein said allodynia is thermalallodynia.
 8. The method according to claim 1, wherein said allodynia iscold allodynia.
 9. The method according to claim 1, wherein saidallodynia is heat allodynia.
 10. The method according to claim 1,wherein said allodynia is mechanical allodynia.
 11. The method accordingto claim 1, wherein the subject to be treated does not experience weightloss.
 12. The method according to claim 1, wherein the subject to betreated is human.
 13. The method according to claim 1, wherein thetreatment is administered by systemic administration.
 14. The methodaccording to claim 1, wherein the treatment is administered byparenteral injection.
 15. The method of claim 14, wherein saidparenteral injection is subcutaneous injection or intrathecal injection.16. The method according to claim 1, wherein the treatment isadministered in dosages of 1 μg/kg-10,000 μg/kg.
 17. The methodaccording to claim 1, wherein said administration is repeated daily. 18.The method according to claim 1, wherein said administration is repeatedat least 1-3 times weekly.
 19. The method of claim 1, wherein saidallodynia is caused by painful diabetic neuropathy, post-herpeticneuralgia, or sciatica
 20. The method according to claim 1, wherein saidtreatment results in substantially full reversal of allodynia in atleast a subset of subjects.
 21. The method according to claim 1, whereinsaid treatment results in disease modification in at least a subset ofsubjects.
 22. A method of treating neuropathic pain in a human subjectin need thereof comprising administering to the subject atherapeutically effective amount of a neurotrophic polypeptidecomprising an amino acid sequence having at least 85% identity to theamino acid sequence of SEQ ID NO: 3, wherein said administration isthree times per week or more infrequently.
 23. The method of claim 22,wherein said administration is weekly or more infrequent administration.24. The method of claim 22, wherein said administration is bi-weekly ormore infrequent administration.
 25. The method of claim 22, wherein thetherapeutic effect of said treatment ameliorates at least one symptom ofneuropathic pain for the entire period between polypeptideadministrations.
 26. The method of claim 25, wherein said symptom isselected from the group consisting of allodynia, hyperalgesia,spontaneous pain, phantom pain, sensations of burning, tingling,electricity, pins and needles, paresthesia, dysesthesia, stiffness,numbness in the extremities, feelings of bodily distortion, andhyperpathia.
 27. The method of claim 22, wherein said treatment does notmaintain measurable levels of said polypeptide in the serum of saidsubject for the entire period between polypeptide administrations. 28.The method of claim 27, wherein the levels of said polypeptide in theserum of said subject falls below 10 ng/mL between polypeptideadministrations.
 29. A method of treating neuropathic pain in a humansubject in need thereof comprising administering to the subject atherapeutically effective amount of a neurotrophic polypeptidecomprising an amino acid sequence having at least 85% identity to theamino acid sequence of SEQ ID NO: 3, wherein said treatment does notmaintain measurable levels of said polypeptide in the serum of saidsubject for the entire interval between polypeptide administrations. 30.The method of claim 29, wherein the levels of said polypeptide in theserum of said subject falls below 10 ng/mL between polypeptideadministrations.